Solid reagent containment unit, in particular for a transportable microfluidic device for sample preparation and molecule analysis

ABSTRACT

A solid reagent containment unit is formed by a support; a frame body fixed to the support and delimiting internally, together with the support, an analysis volume; a reagent-adhesion structure within the analysis volume; and at least one reagent cavity, which extends within the reagent-adhesion structure. The reagent-adhesion structure is of an adhesion material embossable at temperatures lower by 6-8° C. than its own melting point and has a melting point such as not to interfere with the analysis. The reagent cavity forms a retention wall, laterally surrounding the reagent cavity, and houses dried reagents. The adhesion material is chosen among wax, such as paraffin, a polymer, such as polycaprolactone, a solid fat, such as cocoa butter, and a gel, such as hydrogel or organogel.

BACKGROUND Technical Field

The present disclosure relates to a solid reagent containment unit, inparticular for a portable microfluidic device for sample preparation andmolecule analysis. In particular, the present disclosure relates to thefield of so-called Lab-On-a-Chip (LOC) devices, where a singledisposable cartridge (also referred to as disposable unit) comprisesstructures designed to carry out at least some steps of treatment of asample in order to extract and analyze molecules.

Description of the Related Art

In general, disposable cartridges of the above type are put in a machinethat carries out analysis of the substances contained in the cartridge,in general after pre-treatment.

Such systems are of great importance for health, importance thatincreases in time together with the number of analyses that can beperformed in a simple way by a patient alone or with the aid of notparticularly skilled persons.

In particular, the above systems enable analysis of biologicalmolecules, such as nucleic acids, proteins, lipids, polysaccharides,etc. They comprise a plurality of operations that start from the rawmaterial, for example a blood sample. These operations may includevarious degrees of sample pre-treatment the, lysis, purification,amplification, and analysis of the resulting product.

For instance, in DNA-based blood tests, the samples are frequentlypre-treated by filtration, centrifugation, or electrophoresis toeliminate all the non-nucleated cells. Then the remaining white bloodcells are subject to lysis using chemical, thermal, or enzymatic methodsto release the DNA that is to be analyzed. This DNA is then purified, toconcentrate it and eliminate the other molecules in the cells.

Next, DNA is amplified by an amplification reaction, such as PCR(Polymerase Chain Reaction), LCR (Ligase Chain Reaction), SDA(Strand-Displacement Amplification), TMA (Transcription-MediatedAmplification), RCA (Rolling-Circle Amplification), LAMP (Loop-MediatedIsothermal Amplification) and the like.

The procedures are similar if RNA is to be analyzed, but more emphasisis laid on purification to protect the RNA molecule, which is labile.The RNA is usually copied into DNA (cDNA), and then the analysisproceeds as described for DNA.

Finally, the product of amplification undergoes an analysis, usuallybased upon the sequence or dimensions or a combination of both. In ananalysis by hybridization, for example, amplified DNA is passed over aplurality of detectors formed by individual oligonucleotide probes,which are anchored, for example, on electrodes. If the amplified-DNAstrands are complementary to the probes, stable bonds are formed betweenthem, and this hybridization may be read by observing it using a widerange of methods, which include optical or electrical methods.

Other biological molecules are analyzed in a similar way, but typicallypurification is not followed by amplification, and the detection methodsvary as a function of the molecule that is detected. For instance, acommon diagnostic system comprises detection of a specific protein bygetting it to bind to its antibody or using a specific enzymaticreaction. Lipids, carbohydrates, pharmaceuticals, and small moleculescontained in biological fluids are treated in a similar way.

Furthermore, these systems may be used also for the purification ofnon-biological samples, such as water samples, and for the analysis ofnon-biological molecules.

The discussion is here simplified by focusing on purification andanalysis of nucleic acids (DNA and RNA) as example of molecules that canbe purified and analyzed using the cartridge that is the subject of thedisclosure. However, in general, the present cartridge may be used forany chemical or biological test that has the requisites referred tohereinafter.

As regards the purification step, the treatment is based upon thefollowing passages:

movement and mixing of liquid reagents; and

specific capture of the target molecule to be purified usingappropriately functionalized magnetic beads.

As regards the analysis step, the treatment is based upon the followingelements:

thermal control (even be very precise); and

detection using optical methods, such as, purely by way of non-limitingexample, fluorescence or chemiluminescence.

Currently, LOC systems for analysis of nucleic acids have two mainapplications in the field of human diagnostics: quantitative detectionof micro-organisms that cause infective diseases, based uponquantification of nucleic acids of the pathogens;

and detection of specific short subsections within a human genome, whichenables correlation with specific conditions, such as the individualresponse to pharmaceuticals or the predisposition to illnesses. In theformer case, these systems are designed for monitoring health, in stableor emergency conditions (for example, in the case of spread ofepidemics). The latter application regards, among the various contexts,prevention of pathological states and molecular medicine and isincreasing in value over time, since the research in progress findsincreasing correlations between the DNA/RNA sequences and theirfunctions. As a whole, the market for the two applications is expectedto exceed some ten billion dollars in the next few years.

Current systems for analysis of nucleic acids are usually based upon thePCR procedure. This step is typically used in order to obtain asufficient amount of target nucleic acids to be analyzed even startingfrom small samples of biological material. PCR moreover enablessimplification and reduction of the operations of purification of thenucleic acids to be examined since the useful amplified materialconsiderably exceeds the starting material, as well as possible material(such as non-nucleated cells) not useful for analysis.

Execution of PCR typically employs a specific prior preparation of thebiological samples in order to concentrate the nucleic acids, increasingthe sensitivity, and to eliminate substances in the biological samplesthat would inhibit PCR.

With the technique referred to as real-time PCR, PCR is monitored inreal time during amplification, and this enables quantification of thestrands of target nucleic acids based upon amplification curves. To thisend, for example, the material is amplified in presence ofoligonucleotide probes labelled in various ways. If the strands of theamplified target nucleic acids are complementary to the oligonucleotideprobes, in specific conditions of temperature a stable bond is formedbetween them (hybridization). The hybridized material may be detected invarious ways, for example in an optical or electrochemical way.

Lab-On-a-Chip devices are very promising for performing PCR or real-timePCR, in particular in order to obtain fast, automated, and inexpensivetests even in non-hospital environments. However, many current systemsload the cartridge with samples already treated (for example, withDNA/RNA already extracted from the biological sample). This causes theanalysis operations to be more complex due to preliminary treatments,which frequently are done by specialized persons.

It is noted that the ensuing discussion regards purification of nucleicacids and their detection through real-time PCR amplification, asexample of use of the present system. However, the present disclosuremay be applied to other chemical or biological tests.

BRIEF SUMMARY

One or more embodiments of the present disclosure provide solutions thatsimplify treatment and analysis of samples.

According to the present disclosure, a solid reagent containment unit, aportable microfluidic device, a process for manufacturing a solidreagent containment unit, and a method for performing molecule analysesare provided.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

For a better understanding of the present disclosure some embodimentsthereof are now described, purely by way of non-limiting example, withreference to the attached drawings, wherein:

FIG. 1 shows a block diagram of an embodiment of a system for preparingsamples for molecule analysis;

FIG. 2 shows a simplified structural diagram of a control machinebelonging to the system of FIG. 1 ;

FIG. 3A is a perspective view, with parts removed for reasons ofclarity, of an embodiment of the machine of FIG. 2 ;

FIG. 3B is a perspective view similar to FIG. 3A, after insertion of acartridge, showing other parts of the machine;

FIGS. 4, 4A, 5, and 6 show, respectively, an exploded view, aperspective view, a front view, and a ghost back view of a firstembodiment of a cartridge, which may be used in the system for samplepreparation and analysis of FIG. 1 ;

FIGS. 7-10 show the circulation of fluids in successive operative stepsof the cartridge of FIGS. 4-6 ;

FIG. 11 shows a block diagram of another embodiment of a system forsample preparation and analysis;

FIG. 12 shows a simplified structural diagram of a control machinebelonging to the system of FIG. 11 ;

FIGS. 13-15 show, respectively, an exploded view, a front view, and aghost back view of a second embodiment of a cartridge, which may be usedin the system for sample preparation and analysis of FIG. 11 ;

FIGS. 16, 17A, 18A, and 19A show the circulation of fluids on a firstface of the cartridge of FIGS. 13-15 , in successive operative steps;

FIGS. 17B, 18B, and 19B show the circulation of fluids on a second faceof the cartridge of FIGS. 13-15 , in successive operative steps;

FIGS. 20A and 20B are schematic illustrations of the structure of amicrofluidic valve, in the closed condition and in the open condition,respectively, according to one aspect of the present disclosure;

FIGS. 21A and 21B are schematic illustrations of a different embodimentof a microfluidic valve in the closed condition and in the opencondition, respectively;

FIG. 22 is a schematic illustration of another embodiment of amicrofluidic valve, in the closed condition (dashed line) and in theopen condition (solid line);

FIGS. 23A and 23B are schematic illustrations of yet another embodimentof a microfluidic valve in the closed condition and in the opencondition, respectively;

FIGS. 24 and 25 are schematic illustrations of two variants forcontrolling the microfluidic valves of FIGS. 20-23 in the systems ofFIGS. 1 and 11 ;

FIG. 26 is a cross-section of a connector group, according to one aspectof the present disclosure;

FIG. 27 shows a top plan view, with parts in ghost view, of a part ofthe connector group of FIG. 26 ;

FIG. 28 is a cross-section of an assembling step of the connector groupof FIGS. 26-27 ;

FIG. 29 shows the microfluidic connector group of FIG. 26 in a usecondition;

FIG. 30 shows the microfluidic connector group of FIGS. 26-29 applied tothe cartridge 2′ of FIGS. 13-15 ;

FIG. 31 shows a cross-section, exploded view of a container forcollecting samples, according to one aspect of the present disclosure;

FIGS. 32A and 32B show steps of fixing the container of FIG. 31 in asupport;

FIGS. 33-35 show various embodiments of the container of FIG. 31 ;

FIG. 36 shows the container of FIG. 31 applied to the cartridge 2 or 2′of FIG. 5-7 or 13-15 ;

FIG. 37 shows the container of FIG. 31 applied to a variant of thecartridge 2 or 2′ of FIG. 5-7 or 13-15 ;

FIGS. 38A and 38B show cross-sections of a valve group comprising amagnetic valve that may be controllably opened and closed, according toone aspect of the present disclosure, in the closed condition and in theopen condition, respectively;

FIGS. 39A and 39B are perspective views from above and from below,respectively, of a part of the magnetic valve of FIG. 38A, in theundeformed condition;

FIG. 40 is a perspective view from below of the magnetic valve of FIG.38B, in the deformed condition;

FIGS. 41-43 show variants of the magnetic valve of FIGS. 38-40 ;

FIG. 44 shows the magnetic valve of FIGS. 40-42 applied to the cartridge2′ of FIGS. 13-15 ;

FIG. 45 is a schematic side view of a system for mixing liquids in amicrofluidic device;

FIGS. 46A and 46B are front views of a portion of the system of FIG. 45in two different operative steps;

FIG. 47 shows the mixing system of FIG. 45 applied to the cartridge 2 ofFIGS. 5-7 ;

FIGS. 48A and 48B schematically show a cross-section of a microfluidicdevice during two operative steps;

FIG. 49 shows the mixing system of FIGS. 48A and 48B applied to thecartridge 2′ of FIGS. 13-15 ;

FIGS. 50A-50D are cross-sections of a solid-reagent containment unit,according to a further aspect of the present disclosure, in successivemanufacturing steps;

FIG. 50E is a perspective view of the unit of FIG. 50D;

FIG. 51 is a cross-section of the solid-reagent containment unit ofFIGS. 50A-50D in a possible subsequent manufacturing step;

FIGS. 52A-52B are cross-sections of a variant embodiment of thesolid-reagent containment unit in two successive manufacturing steps;

FIGS. 53A-53C are perspective views of different embossing tools used inthe steps of FIGS. 50B and 52A;

FIGS. 54A and 54B are cross-sections of another embodiment of asolid-reagent containment unit, in successive manufacturing steps;

FIG. 55 is a perspective view of an embossing tool used for forming theunit of FIGS. 54A and 54B;

FIGS. 56A-56D are cross-sections of a different embodiment of asolid-reagent containment unit, in successive manufacturing steps;

FIGS. 57A-57C are cross-sections of the solid-reagent containment unitof FIG. 52A in an operative step of a microfluidic device;

FIG. 58 is a perspective view of a different solid-reagent containmentunit, which may be applied to the cartridge 2 or 2′ of FIGS. 4-6 and13-15 , respectively;

FIG. 59 shows the containment unit of FIG. 58 applied to a part of thecartridge 2′ of FIGS. 13-15 ;

FIG. 60 is a top plan view of a different embodiment of the containmentunit of FIG. 58 , which may be applied to the cartridge 2 or 2′ of FIGS.4-6 and 13-15 , respectively;

FIG. 61 is a perspective view of an embossing tool used for forming thecontainment unit of FIG. 60 ;

FIG. 62 is a perspective view of the containment unit of FIG. 60 ;

FIG. 63 is a perspective view of the containment unit of FIG. 62 appliedto a variant of the cartridge 2 or 2′ of FIGS. 4-6 and 13-15 ;

FIG. 64 is a rear view of a part of the cartridge of FIG. 63 ;

FIGS. 65A-65D are schematic illustrations of an analysis unit accordingto yet another aspect of the present disclosure, in successive fillingsteps;

FIGS. 66A-66D show schematically a different analysis unit, insuccessive filling steps;

FIG. 67 shows a variant of the analysis unit of FIGS. 65 and 66 ;

FIG. 68 shows schematically a communication mode used in the presentsystem and toward the outside world;

FIG. 69 is a flowchart of communications between the control machine andthe cartridge in the present system; and

FIG. 70 is a flowchart of communications between the cartridge and anexternal device.

DETAILED DESCRIPTION

The following description relates to a miniaturized (on-chip) cartridge,wherein automated extraction of molecules, in particular nucleic acids,is carried out from a biological sample for their analysis. The systemimplements all the steps envisaged to this end, from loading abiological sample to extracting nucleic acids and collecting them in acollector to enable analysis. The collector may be formed by an analysischamber, where the nucleic acids may be subject to amplification (wherenecessary) and detection, for example using real-time PCR. The structureis such that the movement of the liquids (sample, reagents, and productsof extraction) is obtained by exploiting the force of gravity and asuction pressure generated by an external pump.

FIG. 1 shows a block diagram of an embodiment of a system 1 for thepreparation of biological samples by extracting nucleic acids.

The system 1 comprises a disposable element 2, also referred tohereinafter as cartridge 2, and a control machine 3.

The cartridge 2 comprises a casing 5, having a generallyparallelepipedal shape, housing an extraction chamber 6, a waste chamber7, and a collector 8. In the embodiment described, the collector 8contains assay reagents and forms an analysis chamber, for example anamplification chamber, also designated hereinafter by 8. The chambers 6,7, and 8 have respective vent openings 6A, 7A, 8A and are connectedtogether and to the outside world through a fluidic circuit 9, allowingintroduction of a sample and preparation reagents into the extractionchamber 6, transfer of the treated sample from the extraction chamber 6to the analysis chamber 8, as well as collection of waste material inthe waste chamber 7. In addition, the fluidic circuit 9 enables an airflow from the inlet to the extraction chamber 6, as well as applicationof a suction pressure generated by the control machine 3 in the chambers6-8 in order to transfer the treated sample, the preparation reagents,and the extracted products in the cartridge 2, as described in detailhereinafter with reference to FIGS. 7-10 .

To this end, the cartridge 2 has a sample inlet 10, arranged on a topface 2C of the cartridge 2 (see also FIG. 4A), a fluidic inlet 11 and afluidic outlet 12, arranged on a bottom face 2D of the cartridge 2 (seealso FIG. 4A). The fluidic inlet 11 and the fluidic outlet 12 areconnected to the control machine 3 through a first and a secondconnection element 30A, 30B, arranged on the control machine 3 andforming, together with the respective fluidic inlet 11 and fluidicoutlet 12, a connector group (see also FIGS. 2, 3 and 26-30 ). In FIG. 1(where the connections of a pneumatic type are represented with a thinsolid line, the connections for the liquids are represented with a thicksolid line, and the electrical connections are represented with a dashedand dotted line), the sample inlet 10 is directly connected to theextraction chamber 6, the fluidic inlet 11 (where both air and liquidspass) is connected to the extraction chamber 6 through an inlet channel13, and the fluidic outlet 12 is connected to the vent opening 7A of thewaste chamber 7 through a vent channel 14.

The fluidic circuit 9 of the cartridge 2 further comprises a firstpneumatic channel 16, extending between the vent opening 6A of theextraction chamber 6 and the waste chamber 7 and having a first valve20; a reagent-discharge channel 15, extending between the bottom end ofthe extraction chamber 6 and an intermediate portion of the wastechamber 7 and having a second valve 21; a product-transfer channel 19,extending between the bottom end of the extraction chamber 6 and theanalysis chamber 8; and a second pneumatic channel 17, extending betweenthe vent opening 8A of the analysis chamber 8 and the fluidic outlet 12and having a third valve 22. Alternatively, the second pneumatic channel17 may also be connected to the vent channel 14.

The control machine 3 comprises a pump 25, connected to the fluidicoutlet 12 of the cartridge 2 through a pneumatic duct 55 and to aventilation outlet 26 for generating the suction pressure within thecartridge 2; an actuator group 27, facing the cartridge 2, as describedhereinafter; a supporting structure 28 for the cartridge 2; theconnection elements 30A, 30B, which may be coupled, respectively, to thefluidic inlet 11 and to the fluidic outlet 12 of the cartridge 2; aventilation inlet 33A, connected to the first connection element 30Athrough a ventilation line 33 and having a ventilation valve 34 ; acontrol unit 35, electrically connected to all the members of thecontrol machine 3; and a memory 36, connected to the control unit 35.The control machine 3 may moreover comprise an optical-detection unit37, for detecting the reactions in the cartridge 2, and aheating-control and temperature-monitoring unit 38, for controlling thetemperature (when necessary, for example by carrying out thermal cycles)during analysis of the treated sample, as described in detail below. Airfilters (not shown) may be provided on the ventilation line 33, on theventilation outlet 26, and on the pneumatic duct 55.

The pump 25 is, for example, of a peristaltic, piezoelectric, syringe,or membrane type, or the like, and generates a suction pressure in theregion of 0.05-0.4 atm, for example 0.1 atm in the case of a peristalticpump and 0.4 atm in the case of a membrane pump.

The actuator group 27 comprises one or more magnetic-valve actuators 40,facing the cartridge 2 for controlling the valves 20-22, as described ingreater detail hereinafter with reference to FIGS. 2, 3A, and 3B; ananchor actuator 41, facing the analysis chamber 8, for controlling themovement of a mixing anchor, as described in greater detail hereinafterwith reference to FIG. 9 ; cooling fans 42, also facing the cartridge 2for reducing the temperature on the basis of any thermal cycles duringanalysis of the treated sample; and a blocking actuator 43, for trappingmagnetic particles, for example magnetic beads, during samplepreparation, as described in greater detail hereinafter with referenceto FIG. 9 .

The control machine 3 moreover carries a reagent-supporting structure45, accommodating a plurality of containers 46 (see also FIGS. 2 and 3A)and connected to the first connection element 30A.

A heating and temperature-control element 48 is coupled to the analysischamber 8 and is controlled by the heating-control unit 38 through anelectrical-connection element 47. For instance, as described in greaterdetail hereinafter (FIGS. 4-6 ), the heating and temperature-controlelement 48 may be a silicon chip housing integrated resistors.

FIGS. 2, 3A, and 3B show in a simplified way the structure of thecontrol machine 3. In detail, the control machine 3 comprises a base 50closed at the top by a manifold structure 51 and housing aprinted-circuit board 52 (FIGS. 3A and 3B), which carries the controland processing unit 35 and the heating-control unit 38 (FIG. 2 ).Moreover, the base 50 accommodates the pump 25; reagent valves 53connected to the reagent containers 46; a fluidic duct 54, connectingthe reagent valves 53 to the first connection element 30A; and thepneumatic duct 55, connecting the pump 25 to the second connectionelement 30B.

The connection elements 30A and 30B are carried by the manifoldstructure 51 and each comprise a needle 58, designed to be inserted inthe cartridge 2 and to perforate respective gaskets 120 (FIG. 4 ) in thecartridge 2, as described in greater detail hereinafter with referenceto FIGS. 4-6 ).

The supporting structure 28 is carried by the manifold structure 51 andcomprises two U-shaped guides 61 defining mutually facing grooves 62,for allowing insertion of the cartridge 2. The supporting structure 28further comprises a horizontal bar 63, extending between the guides 61,and from which the needles 58 project upwards. The needles 58 of theconnection elements 30A and 30B thus automatically penetrate into thefluidic inlet 11 and the fluidic outlet 12 of the cartridge 2 when thecartridge is inserted in the grooves 62 and pushed down until it restsagainst the horizontal bar 63.

Furthermore, the manifold structure 51 carries the magnetic valveactuator 40, the anchor actuator 41, the optical-detection unit 37, theblocking actuator 43 (adjacent to the supporting structure 28 so as toface the cartridge 2, when the latter is inserted in the supportingstructure 28), as well as the cooling fan 42.

In detail, the magnetic valve actuator 40 comprises a first turret 64carrying a first magnetic element 70 (for example, a permanent magnet)mobile along the first turret 64. The first turret 64 may be a wormscrew rotated about a vertical axis (perpendicular to the manifoldstructure 51) by a first electric motor 71 in the base 50 (FIG. 2 ),co-operating with a counter-thread formed on a support of the firstmagnet or of the first magnetic element 70 so as to be brought each timeinto a facing position with one of the valves 20-22, according to theoperative step of the system 1. Alternatively, the magnetic valveactuator 40 may comprise a plurality of electromagnets, one for eachvalve 20-22 of the cartridge 2, fixed on the first turret 64 (which, inthis case, is formed by a simple supporting vertical structure), facinga respective valve 20-22, and selectively operated when desired (asexplained in greater detail hereinafter with reference to FIGS. 7-10 ).

The anchor actuator 41 comprises a second turret 65, carrying a secondmagnetic element 73 (for example, a permanent magnet) mobile along thesecond turret 65 and rotatable about a horizontal axis. In particular,the second magnetic element is mobile along the height of the extractionchamber 6 and governs displacement and rotation of an anchor 97 (FIG. 4) in the extraction chamber 6, for stirring and mixing the liquidpresent therein, as explained with reference to FIGS. 9 and 45-47 . Forinstance, the second turret 65 may also be a worm screw, extendingvertically, rotatably driven by a second electric motor 72 (alsoarranged in the base 50, FIG. 2 ) and co-operating with a counter-threadformed on the casing of a third electric motor 75 (FIG. 3A). The thirdmotor 75 is horizontally rotatable and carries the second magneticelement 73, which can thus translate vertically and rotate.

The blocking actuator 43 comprises an arm 77 carrying a permanent magnet78. The arm 77 is brought to and away from the cartridge 2 by a fourthelectric motor 79, fixed to the manifold structure 51. Alternatively,the support 77 may be fixed, and the permanent magnet 78 can be replacedby an electromagnet that is activated/deactivated according to theoperative step of sample preparation, as explained in detail hereinafterwith reference to FIG. 9 .

The optical-detection unit 37 (FIG. 3B), carried by the base 50 througha support 76, is arranged alongside the supporting structure 28, on theopposite side of the cartridge 2 (after insertion of the latter in theguides 61) with respect to the turrets 64-65. The optical-detection unit37 has the function of detecting the reaction that is occurring (or hasoccurred) in the analysis chamber 8, for example by optical detection ofnucleotide fragments hybridized to corresponding labelled detectionfragments, in a per se known manner.

FIGS. 4-6 show a possible implementation of the structure of a cartridge2 that can be used with the system 1 of FIGS. 1-3 . In detail (FIG. 4A),the cartridge 2 has a generally parallelepipedal shape configured to bemounted in the control machine 3 so that its major dimension (hereafterdenoted as height H) is arranged vertically. In addition, the cartridge2 has another of the three dimensions (referred to as thickness T) muchsmaller than the other two dimensions, and the third dimension, referredto as width L, with an intermediate value. For instance, the cartridge 2may have H=75 mm, L=50 mm, and T=10 mm.

The cartridge 2 is delimited by a front face 2A, a back face 2B, a topface 2C, a bottom face 2D, a first lateral face 2E, and a second lateralface 2F (where “top” and “bottom” refer to the position of in thecontrol machine 3). The lateral faces 2E, 2F are designed to be insertedin the guides 61, with the front face 2A facing the optical detector 37and the back face 4B facing the turrets 64 and 65. The bottom face 2D isdesigned to be introduced in the supporting structure 28 first and toarranged against the horizontal bar 63 of the control machine 3 (FIGS. 2and 3 ).

The cartridge 2 is here formed by three parts, all of transparentmaterial: a body 80; a first closing wall 81; and a second closing wall82, for example a film. The parts 80-82 are bonded together, for exampleglued or welded thermally, and may have gaskets and sealing means (notshown) to prevent leakage of liquids towards the outside, and to ensureseparation of the various channels from each other and isolation fromthe external environment.

The body 80, for example of molded plastic, has a first main face 80Aand a second main face 80B, opposite to each other; and a top face 80Cand a bottom face 80D, opposite to each other, forming in part the topface 2C and the bottom face 2D of the cartridge 2. The first closingwall 81 has a face 81A fixed to the second main face 80B of the body 80;the second closing wall 82 is bonded to the first face 80A of the body80.

The first and second main faces 80A, 80B of the body 80 are shown indetail in FIGS. 5 and 6 . The first main face 80A has a plurality ofrecesses and openings that form, together with other recesses andopenings on the face 81A of the first closing wall 81, the chambers 6-8of FIG. 1 and the channels 14-19. Moreover, the body 80 has a first, asecond and a third valve hole 90-92, where the valves 20-22 of FIG. 1are formed, as described in detail hereinafter. To clarify further thestructure, in FIGS. 5 and 6 recesses and cavities formed on the firstface 81A of the closing wall 81 are moreover represented dashed.

In detail, the first main face 80A of the body 80 has a first recess(referred to hereinafter as extraction recess 83 since it forms,together with the second closing wall 82, the extraction chamber 6) anda second recess (referred to hereinafter as analysis recess 8 since itforms the analysis chamber 8, as explained hereinafter). The body 80moreover has a through opening 85 since it forms, together with theclosing walls 81, 82, the waste chamber 7, as explained hereinafter.

The extraction recess 83 is generally V-shaped with its bottom portionnear the bottom face 80D and its top portion, wider than the bottomportion, near the top face 80C. Thus, by virtue of the use arrangementof the cartridge 2, the extraction chamber 6 has a vertical maindimension, narrower at the bottom and wider at the top. In particular,the extraction recess 83 has an aspect ratio (the ratio between thevertical, larger dimension and the smaller, horizontal dimension) of atleast 5, typically approximately 10.

Moreover, the extraction recess 83 accommodates the anchor 97 and atablet 98 containing the magnetic beads that capture the nucleic acids.In a per se known manner, the tablet 98 may be produced by oven dryingor lyophilizing a solution containing the magnetic beads.

The analysis recess 84 has a generally parallelepipedal shape, here likea bag, with bottom rounded corners, and is closed at the sides by achip, which forms the heating and temperature-control element 48 and isthus designated by 48. The chip 48 is inserted in a through opening 84A(FIG. 4 ) having a parallelepipedal shape, formed in the second closingwall 82 and sealed in any suitable way. For instance, the chip 48 may beglued to the first main face 80A of the body 80. In practice, the chip48 and the analysis recess 84 form the collector (analysis chamber) 8.The chip 48 has on the back (on the face 2B of the cartridge 2, FIG. 4A)electrical contacts 49 designed to be electrically coupled with theelectrical-connection element 47 on the control machine 3 (FIG. 1 ).

It is noted that, if the collector 8 is limited to collecting theseparated nucleic acids and does not contain assay reagents, thecartridge 2 may be equipped with a further fluidic outlet (not shown),closed, for example, by a perforable gasket, allowing recovery of thetreated nucleic acids, for example, using a syringe. In this case, thechip 48 may be missing.

An introduction opening 117 extends from the top face 80C of the body 80to the extraction recess 83 to form the sample inlet 10 of FIG. 1 . Theintroduction opening 117 may be closed by a plug element 89, representedschematically in FIGS. 4 and 5 . Furthermore, the introduction opening117 may have a screw portion (not shown), for screwing a samplecontainer (not shown either), and/or may have reclosing means, forexample as described in detail hereinafter with reference to FIGS. 26-30. A lateral opening 118 may be arranged alongside the introductionopening 117 to enable venting of the extraction recess 83 duringintroduction of a sample into the sample inlet 10.

The bottom portion of the extraction recess 83 is in fluidic connectionwith a coiled inlet fluidic recess 86, which extends on a first side (onthe left in FIG. 5 ) of the extraction chamber 6 from the bottom portionas far as near the top face 80C of the body 80 and then from here as faras near the bottom face 80D, where a first through hole 87 connects thefirst main face 80A to the second main face 80B of the body 80. Inpractice, the inlet fluidic recess 86 forms the inlet channel 13 of FIG.1 . An output fluidic recess 88, also coiled, extends on a second sideof the extraction chamber 6 (on the right in FIG. 5 ), from the bottomportion as far as near the top face 80C of the body 80 and then fromhere up to the second valve hole 91, at an intermediate height withrespect to the cartridge 2. The second valve hole 91 is verticallyaligned (in the in-use position of the cartridge 2) to the first valvehole 90 and to the third valve hole 92, which are arranged,respectively, above the second valve hole 91 (near the top face 80C ofthe body 80) and below the second valve hole 91. The first, second, andthird valve holes 90-92 connect the first main face 80A to the secondmain face 80B of the body 80 and are normally closed by respectiveshutters 140-142 represented in ghost view only in FIG. 4 . Forinstance, the shutters 140-142 are elastic elements that undergodeformation under the action of an external magnetic field, opening therespective valve holes 90-92, as described in greater detail hereinafterwith reference to FIGS. 38-44 , and form, together with the respectivevalve holes 90-92, the valves 20-22 of FIG. 1 .

The first valve hole 90 is fluidically connected to the vent opening 6Aof the extraction recess 83 through a first vent recess 95 formed on thefirst main face 80A, and to an intermediate portion of the waste opening85 through a first L-shaped fluidic recess 96 formed on the face 81A ofthe first closing wall 81 (FIG. 4 ). In practice, the first fluidicrecess 96 has a first portion 96A that extends between the first and thesecond valve holes 90, 91, and a second portion 96B that extends betweenthe second valve hole 91 and the waste recess 85. In practice, the firstvent recess 95 and the first fluidic recess 96 form the first pneumaticchannel 16 of FIG. 1 . In addition, the second portion 96B of the firstfluidic recess 96 forms, together with the output fluidic recess 88, thereagent-discharge channel 15 of FIG. 1 .

The output fluidic recess 88 is moreover connected to the analysisrecess 84 through a product recess 99 (having a first portion 99A, whichextends on the first main face 80A of the body 80, and a second portion99B, which extends on the face 81A of the first closing element 81, FIG.4 ) and a first pair of through holes 100A and 100B, which extend in thebody 80. The first portion 99A extends from the output fluidic recess 88towards the top face 80C of the body 80 and then downwards, towards thebottom face 80D, to prevent the products extracted from the sample to betransferred to the waste chamber 7, as explained hereinafter. Inpractice, the output fluidic recess 88 and the product recess 99 formthe product-transfer channel 19 of FIG. 1 .

The third valve hole 92 is connected to the vent opening 8A of theanalysis recess 84 through a second vent recess 101 and a second pair ofthrough holes 102A, 102B. The second vent recess 101 has a first portion101A extending on the first main face 80A of the body 80 and a secondportion 101B extending on the face 81A of the first closing element 81(FIG. 4 ).

A first cavity 104 (FIG. 4 ) extends on the face 81A of the firstclosing wall 81 from the bottom face 2D of the cartridge 2 up to a firstchamber-like recess 105 facing the first through hole 87 (FIG. 5 ). Asecond cavity 106 extends on the face 81A of the first closing wall 81from the bottom face 2D of the cartridge 2 up to a second chamber-likerecess 107 where ends a second through hole 108 formed in the body 80.The first cavity 104, the first chamber-like recess 105, and the firstthrough hole 87 form the fluidic inlet 11; the second cavity 106, thesecond chamber-like recess 107, and the second through hole 108 form thefluidic outlet 12 of FIG. 1 . Gaskets 120, for example of rubber areinserted in the cavities 105, 107 and hermetically seal the fluidicinlet 11 and the fluidic outlet 12 prior to insertion of the cartridge 2in the control machine 3; the gaskets may be easily be perforated by theneedles 58 (FIGS. 2 and 3 ).

The second through hole 108 places the second chamber-like recess 107 influidic communication with a first end of a third vent recess 110, whichhas a first portion 110A extending on the first main face 80A, partiallyalong the output fluidic recess 88, and a second portion 110B thatextends on the first closing wall 81. A third through hole 113 arrangedin a middle area of the first portion 110A of the third vent recess 110connects the third vent recess 110 to the third valve hole 92 through afourth vent recess 114, which extends on the face 81A of the firstclosing element 81 (FIG. 4 ). The first portion 110A of the third ventrecess 110, the third through hole 113, the fourth vent recess 114, thesecond vent recess 101, and the second pair of through holes 102A, 102B,form the second pneumatic channel 17 of FIG. 1 .

A fourth through hole 115 connects the first portion 110A to the secondportion 110B of the third vent recess 110 (FIG. 4 ); the second portion110B of the third vent recess 110 ends into a waste recess 85A on thefirst closing wall 81. The waste recess 85A on the first closing wall 81is congruent with and faces the waste opening 85 in the body 80 andforms with this, and with the corresponding portion of the secondclosing wall 82, the waste chamber 7 of FIG. 1 , as already mentioned.The area where the second portion 110B of the third vent recess 110 endsinto the waste recess 85A thus forms the vent opening 7A of the wastechamber 7, which is set at the highest point (when the cartridge 2 isinserted in the control machine 3) of the waste chamber 7, to ensurethat the liquid cannot exit from the waste chamber 7. In practice, thesecond portion 110B of the third vent recess 110, the fourth throughhole 115, and the third vent recess 110 form the vent channel 14 of FIG.1 .

The waste opening 85 and the waste recess 85A are sized so that thewaste chamber 7 has a greater volume than all the spent reagentsdischarged therein, as explained hereinafter.

In practice, the recesses, holes, and openings 83-117 in the cartridge 2form the fluidic circuit 9 of FIG. 1 and, as has been mentioned, arearranged so that the displacement of the liquids and the correspondingdisplacement of the air occur by exploiting the force of gravity and aslight suction pressure applied on the fluidic outlet 12 (recesses 106,106A, 107, 107A), as described in detail hereinafter with reference toFIGS. 7-10 . In particular, FIGS. 7-10 show the movement of fluids inthe body 80 in the successive operative steps. For greater clarity,FIGS. 7-10 shows the body 80 in ghost view and the fluidic structures ofthe first closing wall 81, irrespective whether the recesses, openings,and chambers are arranged on the first main face 80A, on the second mainface 80B or on the first closing wall 81.

In detail, the cartridge 2 is inserted in the supporting structure 28 sothat the second closing wall 82 faces the turrets 64-65, and theanalysis chamber 8 (containing the assay reagents, for example,amplification reagents) faces the optical detector 37. The contacts 49on the chip 48 thus are brought at the electrical-connection element 47(FIG. 1 ) on the control machine 3, thus connecting the chip 48 to thecontrol unit 35. During insertion of the cartridge 2, the needles 58perforate the gaskets 120, thus connecting the fluidic inlet 11 and thefluidic outlet 12 to the control machine 3. Then, a liquid sample isintroduced into the extraction chamber 6. The liquid sample isintroduced through the introduction opening 117, for example using asyringe (not shown), which perforates (arrow 150 of FIG. 7 ) the plugelement 89. Alternatively, a container (not shown), as described ingreater detail hereinafter with reference to FIGS. 31-37 , or a plugsuch as for a test tube may be screwed on the introduction opening 117shown. In this step, all the valves 20-22 are closed, and the pump 25(FIG. 1 ) is inactive. The liquid sample accumulates by gravity on thebottom of the extraction chamber 6 and fills it partially (typically,less than half full, for example approximately one fifth). Afterintroducing the liquid sample, if necessary, the introduction opening117 is reclosed.

Next, FIG. 8 , the preparation reagents are introduced into theextraction chamber 6, and the nucleic acids are extracted from theliquid sample in the extraction chamber 6.

To this end, the containers 46 (FIGS. 1-3 ) are selectively connected insequence, according to envisaged procedures, to the fluidic inlet 11through the reagent valves 53 and the first fluidic duct 54 (FIG. 2 ).In this step, the first valve 20 is opened, by causing deformation ofthe first shutter 140 (FIG. 4 ). In this way, the top part of theextraction chamber 6 is connected to the waste chamber 7 through theextraction vent opening 6A, the first vent recess 95, the first valvehole 90, and the first fluidic recess 96, as indicated by the arrow 151.Furthermore, the pump 25 is activated so as to generate a suctionpressure in the waste chamber 7, through the fluidic outlet 12, thesecond chamber-like recess 107, the second through hole 108, the thirdvent recess 110, the fourth through hole 115, the second portion 110B ofthe third vent recess 110, and the vent opening 6A, as indicated by thearrow 152. In this way, the preparation reagents at the fluidic inlet 11are lead, by the suction pressure generated by the pump 25, towards thebottom part of the extraction chamber 6, through the first chamber-likerecess 105 and the inlet fluidic recess 86, as indicated by the arrow153, and the air in the extraction chamber 6 can flow away from the topof the extraction chamber 6 towards the waste chamber 7.

After introducing the first preparation reagent, namely, the lysisliquid, proper lysis is carried out in a per se known manner.

During lysis, the anchor actuator 41 may be operated to cause a repeatedvertical movement and rotation of the anchor 97 (FIG. 4 ) in theextraction chamber 6 in order to stir and mix the lysis liquid (as wellas of the magnetic beads capturing the nucleic acids, as describedhereinafter with reference to FIGS. 45-47 ).

At the end of lysis, spent lysis reagents are discharged into the wastechamber 7 (FIG. 9 ). To this end, the fluidic inlet 11 is connected tothe external environment (by opening the ventilation valve 34 of thecontrol machine 3 and connection to the ventilation inlet 33A of themachine, FIG. 1 ), thus allowing the air to flow into the cartridge 2through the inlet fluidic recess 86 towards the bottom end of theextraction chamber 6 (arrow 154). In addition, the magnetic valveactuator 40 opens the second valve 21 by causing deformation of thesecond shutter 141 and freeing the second valve hole 91. The bottom endof the extraction chamber 6 is thus connected to the waste chamber 7through the output fluidic recess 88 and the second portion 96B of thefirst fluidic recess 96 (arrow 155). Also in this step, the pump 25 isactive and generates a suction pressure in the waste chamber 7, throughthe fluidic outlet 12, the second chamber-like recess 107, the secondthrough hole 108, the third vent recess 110, and the fourth through hole115, as indicated by the arrow 156. In this step, the blocking actuator43 is activated and attracts the magnetic beads coupled to the nucleicacids. The magnetic beads are generally already in the extractionchamber 6, having been introduced in the manufacturing step, and arehere contained in the tablet 98 (FIG. 4 ), which dissolves when thesample or the first lysis liquid is introduced in a per se known manner.The separated nucleic acids are thus held in the extraction chamber 6 bythe magnetic attraction generated by the blocking actuator 43, whereasthe spent lysis reagents are drawn into the waste chamber 7 by theaction of the suction pressure generated by the pump 25 and the force ofgravity. It is noted that, in this step, even though the product recess99 is free, the spent lysis reagents do not pass through it, since thefirst stretch thereof is in a higher position than the second portion96B of the first fluidic recess 96 and as a result of the suctionpressure existing in the waste chamber 7. The air displaced in the wastechamber 7 may be discharged outwards through the fluidic outlet 12.

Next, in a known manner, the nucleic acids are flushed by introducing insequence appropriate flushing liquids supplied by the containers 46(FIG. 1 ), according to the path indicated by the arrow 153 of FIG. 8 ,and subsequently discharging them, along the path indicated by the arrow156 of FIG. 9 , as described previously in detail for the lysis liquid.Also during the flushing step, the anchor actuator 41 may be operated toobtain stirring and mixing of the liquid and the magnetic beads. Inaddition, the fluidic inlet 11 may be connected to the ventilation inlet33A of the control machine 3 (FIG. 1 ) by opening the ventilation valve34, allowing the air to flow into the cartridge 2 towards the bottom endof the extraction chamber 6 through the inlet fluidic recess 86. Asdescribed in detail with reference to FIGS. 48-49 , this enablesbubbling of air in the extraction chamber 6 and re-mixing of the liquidand of the magnetic beads present therein.

Flushing may comprise a number of cycles with different liquids, in aper se known manner.

At the end of this step, only the nucleic acids attached to the magneticbeads are present on the bottom of the extraction chamber 6.

Next, the nucleic acids, by now purified, are eluted via an expresslyprovided elution liquid. In this step, the nucleic acids are separatedfrom the magnetic beads and dispersed in the elution liquid. In thisstep, air may again be bubbled in the extraction chamber 6 to favordetachment, as discussed in greater detail hereinafter with reference toFIGS. 48-49 . Moreover, the anchor actuator 41 may again be operated(FIGS. 2, 3A).

In FIG. 10 , the extracted nucleic acids and the elution liquid are sentto the collector or analysis chamber 8 by the action of the suctionpressure generated by the pump 25 and the force of gravity. In thisstep, the first and second valve holes 90, 91 are closed by thecorresponding shutters 140, 141 (by closing the first and second valves20, 21), and the third valve hole 92 is opened by causing deformation ofthe third shutter 142 (by opening the third valve 22). Thus, the suctionpressure generated by the pump 25 causes suction of the liquid on thebottom of the extraction chamber 6 and of the extracted nucleic acids(which are no longer bound to the magnetic beads) through the outputfluidic recess 88, the product recess 99, and the first pair of throughholes 100A and 100B (arrow 157). In this step, the magnetic beads arewithheld in the extraction chamber 6 by the blocking actuator 43 (FIG. 1). It is noted that, in this step, closing of the second valve hole 91prevents discharge of the extracted nucleic acids into the dischargechamber 7. Since the third valve 22 is open, the suction pressuregenerated by the pump 25 also causes suction and discharge of the air inthe analysis collector/chamber 8 towards the fluidic outlet 12 of thecartridge 2 through the second vent recess 101, the second pair ofthrough holes 102A, 102B, the second valve hole 92, the fourth ventrecess 114, the third through hole 113, third vent recess 110, and thesecond chamber-like recess 107 (arrow 158).

In this step, the fluidic inlet 11 is connected to the externalenvironment and allows the air to flow into the extraction chamber 6 asdescribed above with reference to the arrow 154. Consequently, also inFIG. 10 , the flow of air from the outside towards the extractionchamber 6 is indicated by the arrow 154. The air introduced from thefluidic inlet 11 then rises towards the top part of the extractionchamber 6, facilitating displacement of the liquid present on the bottomof the extraction chamber 6 towards the extraction collector/chamber 8.

The nucleic acids are then transferred into the analysiscollector/chamber 8, from where they may be recovered or whereamplification of the nucleic acids and their analysis may be carried outin a per se known manner.

According to a different embodiment, the cartridge already contains thepreparation reagents used in the extraction chamber 6. In this case, thecontrol machine 3 of FIGS. 1-3B is modified as shown in FIGS. 11-12 ,and the cartridge 2 of FIGS. 4-6 is modified as shown in FIGS. 13-15 .Thus, in FIGS. 11-15 , elements that are the same as the ones describedfor the embodiment of FIGS. 1-6 are designated by the same referencenumbers and the elements that are modified are distinguished by primesigns.

In detail, FIGS. 11 and 12 show a system 1′ comprising a cartridge 2′and a control machine 3′.

The control machine 3′ differs from the control machine 3 of FIGS. 1-3Bin that the actuator group 27′ further comprises a reagent actuator 160facing the cartridge 2′. The reagent actuator 160 is controlled by thecontrol unit 35′.

Furthermore, the control machine 3′ differs from the control machine 3in that it does not carry any reagent-supporting structure (45 in FIG. 1) and thus does not comprise reagent valves and the correspondingfluidic duct (53 and 54 in FIG. 1 ). Thus, in the control machine 3′ thefirst connection element 30A is connected only to the ventilation inlet33A. For the rest, the control machine 3′ may be the same as the controlmachine 3 of FIGS. 1-3 .

As mentioned, the cartridge 2′ contains the preparation reagents usedfor extracting the nucleic acids. To this end, the cartridge 2′comprises a plurality of reagent chambers 165 arranged on the secondmain face 80A′ of the body 80′ (FIG. 15 ), connected to the fluidicinlet 11 through respective reagent holes 167 traversing the body 80′and closed by one-shot valves 168 (FIG. 13 ). The one-shot valves 168are controlled by the reagent actuator 160. For instance, the one-shotvalves 168 may be plugs of wax or any other heat-meltable material, asdescribed in detail hereinafter with reference to FIGS. 20-25 . In thiscase, the reagent actuator 160 may comprise a plurality of meltingelements 161 (FIG. 12 ), for example a plurality of LEDs that, whenactivated, cause melting of the material of the one-shot valves 168 andopening of the corresponding reagent holes 167. In FIG. 12 , the meltingelements 161 are arranged on a turret 166 and connected to the controlunit 35 controlling turning-on and -off of the melting elements 161.

As an alternative to the above, the reagent actuator 160 on the controlmachine 3′ may comprise just one melting element 161, which can bedisplaced to a position facing the one-shot valve 168 to be operatedeach time, using a motor-and-worm mechanism similar to that of themagnetic valve actuator 40.

FIGS. 13-15 show an embodiment of the structure of the cartridge 2′housing the reagent chambers 165. As may be noted, the reagent chambers165 are arranged on the second main face 80B′ of the body 80′,approximately behind the extraction chamber 6, are closed at the back bythe first closing wall 81′, are arranged contiguous to each other, areL-shaped, and are separated from each other by container walls 169. Thereagent chambers 165 have respective ends, where the reagent holes 167open. The reagent holes 167 are thus arranged vertically aligned to theinlet fluidic recess 86 and are closed by the one-shot valves 168 so asto separate the reagent chambers 165 from the inlet fluidic recess 86when the one-shot valves 168 are closed and to be connected to the inletfluidic recess 86 when the respective one-shot valves 168 are opened.

In the embodiment of FIGS. 13-15 , the fluidic circuit 9′ of thecartridge 2′ is slightly modified with respect to what described withreference to the cartridge 2 of FIGS. 4-6 . In particular, the fluidiccircuit 9′ is formed by recesses, cavities, and holes, all extending inthe body 80′ and closed on one side by the first closing wall 81′ or thesecond closing wall 81′, 82′ according to whether they are arranged onthe first main face 80A′ or the second main face 80B′ of the body 80.

Here (FIG. 14 ), the fluidic inlet 11 is formed by a first blind hole170 (represented dashed), which extends from the bottom surface 80D′ andis connected to the inlet fluidic recess 86 through an inlet hole 171.The fluidic outlet 12 is here formed by a second blind hole 172 (alsorepresented dashed), which extends from the bottom surface 80D′ and isconnected to the third vent recess 110′ through a first communicationhole 175. In turn, the third vent recess 110′, similar to the third ventrecess 110 of the cartridge 2, extends vertically on the first main face80A′ of the body 80, here in proximity of the second main face 80F′, andis connected in an intermediate portion thereof to the third valve hole92′ through the third through hole 113′ and the fourth vent recess 114′formed on the second main surface 80B′ of the body 80′ (FIG. 15 ). Inaddition, the third vent recess 110′ is connected, near its top end, toa second communication hole 178, which connects the first main face 80A′to the second main face 80B′ of the body 80′, at the waste chamber 7′.

The waste chamber 7′ (FIG. 15 ) is formed by a waste recess 85′extending over a large part of the second main face 80B′ of the body80′, along the area of the reagent chambers 165, and in fluidiccommunication with the first and second valve holes 90, 91. A firstpartition wall 180 separates the waste chamber 7′ from the fourth ventrecess 114′ (and thus from the third valve hole 92), and a secondpartition wall 181 separates the waste chamber 7′ from the analysisrecess 84′ (formed on the first main face 80A′, FIG. 14 ). The analysisrecess 84′ here has a parallelepipedal shape connected to an analysisopening 84B extending through the body 80′.

Here, the product recess 99′ and the second vent recess 101′ are formedcompletely on the first main face 80A′ (FIG. 15 ) and are in fluidicconnection with the analysis recess 84′ through a fourth communicationhole 100′ and a fifth communication hole 102′, respectively, and theanalysis opening 84B (FIGS. 14 and 15 ).

Operation of the system 1′ will be described hereinafter with referenceto FIGS. 16-19B.

In detail, the cartridge 2′ is inserted in the supporting structure 28so that the second, covering, wall 82′ faces the turrets 64-65 and theanalysis chamber 8′ (containing the assay reagents) faces the opticaldetector 37. The contacts 49 on the chip 48 thus each a position facingthe electrical-connection element 47 (FIG. 11 ) on the control machine3′, thus connecting the chip 48 to the control unit 35.

Also in this case, during insertion of the cartridge 2′, the needles 58perforate the gaskets (not shown since they are similar to the gaskets120 of FIG. 4 ) in the blind holes 170, 172, thus connecting the fluidicinlet 11′ and the fluidic outlet 12′ to the control machine 3′.

Next (FIG. 16 ), a liquid sample is introduced into the extractionchamber 6 (arrow 200 of FIG. 16 ) by using a syringe or by screwing acontainer (not shown), as described in greater detail hereinafter withreference to FIGS. 31-37 . In this step, all the valves 20-22 areclosed, and the pump 25 (FIG. 11 ) is inactive.

Then (FIGS. 17A and 17B), the preparation reagents are introduced intothe extraction chamber 6. To this end, the one-shot valves 168 areopened in the sequence provided for by the respective reagent actuators160 (FIG. 11 ); for example, they are melted by the respective LEDs 161,as explained in detail with reference to FIGS. 20-25 . Opening of eachone-shot valve (in FIG. 17A, see the one-shot valve designated by 168A),and thus freeing of the respective hole 167, enables the liquid in thecontainer 165 associated to the one-shot valve 168A to flow towards theinlet fluidic recess 86 and from there into the extraction chamber 6,towards its bottom end (arrow 201). Introduction of the lysis reagentson the bottom of the extraction chamber 6 is thus favored by the forceof gravity and by the presence of the suction pressure generated by thepump 25.

In this step, the first valve hole 90 is opened by causing deformationof the first shutter 140, thus connecting the top part of the extractionchamber 6 to the waste chamber 7′ (arrow 203). Furthermore, the pump 25is activated so as to generate a suction pressure in the waste chamber7′ through the fluidic outlet 12, the second blind hole 172, the firstcommunication hole 175, the third vent recess 110′, and the secondcommunication hole 178 (arrow 202), also favoring discharge of air fromthe top part of the extraction chamber 6 (arrow 203).

After introducing the first preparation reagent, namely, the lysisliquid, proper lysis is carried out in a per se known manner.

During lysis, the anchor actuator 41 may be operated to cause a repeatedvertical movement and rotation of the anchor 97 (FIG. 4 ) in theextraction chamber 6 in order to stir and mix the lysis liquid and themagnetic beads capturing the nucleic acids, as described with referenceto FIGS. 45-47 .

At the end of lysis, the spent lysis reagents are discharged into thewaste chamber 7′ (FIGS. 18A and 18B). To this end, the fluidic inlet 11is connected to the external environment (by opening the ventilationvalve 34 of the control machine 3′ and connection to the ventilationinlet 33A — FIG. 11 ), allowing air to flow into the cartridge 2′towards the bottom end of the extraction chamber 6 through the firstblind hole 170, the inlet hole 171, and the inlet fluidic recess 86(arrow 204). Furthermore, the magnetic valve actuator 40 (FIG. 11 )opens the second valve 21 by causing deformation of the second shutter141, thus freeing the second valve hole 91. The bottom end of theextraction chamber 6 is thus connected to the waste chamber 7′ throughthe output fluidic recess 88 (arrow 205). Also in this step, the pump 25is active and generates a suction pressure in the waste chamber 7′,through the fluidic outlet 12, the second blind hole 172, the firstcommunication hole 175, the third vent recess 110′, and the secondcommunication hole 178, as indicated by arrow 206. In this step, theblocking actuator 43 is activated and attracts the magnetic beadscoupled to the nucleic acids. Also in this case, the magnetic beads mayalready be contained in the extraction chamber 6. The nucleic acids arethus withheld in the extraction chamber 6 by the magnetic attractiongenerated by the blocking actuator 43, whereas the spent lysis reagentsare drawn into the waste chamber 7′ by the action of the suctionpressure generated by the pump 25 and the force of gravity. The airdisplaced into the waste chamber 7′ may be discharged towards theoutside through the fluidic outlet 12.

Then, in a known manner, the nucleic acids are flushed by introducing insequence appropriate flushing liquids contained in the reagent chambers165 according to what described with reference to FIGS. 18A and 18B, andby subsequently discharging them, along the path indicated by arrow 205of FIGS. 18A and 18B, as described previously in detail for the lysisliquid. During flushing, the anchor actuator 41 may be operated forstirring and mixing the liquid and the magnetic beads. Furthermore, thefluidic inlet 11 may be connected to the ventilation inlet 33A of thecontrol machine 3′ (FIG. 12 ) by opening the ventilation valve 34, thusallowing air to flow into the cartridge 2′ towards the bottom end of theextraction chamber 6 through the inlet fluidic recess 86, enabling airbubbling in the extraction chamber 6 and remixing of the liquid and ofthe magnetic beads therein.

Flushing may comprise a number of cycles with different liquids, in aper se known manner.

At the end of this step, only the nucleic acids attached to the magneticbeads step remain on the bottom of the extraction chamber 6.

Next, the nucleic acids are eluted using a suitable elution liquid. Inthis step, the now purified nucleic acids are separated from themagnetic beads and dispersed in the elution liquid. In this step, airmay be bubbled again in the extraction chamber 6 to favour detachment,as discussed in greater detail hereinafter with reference to FIGS. 48-49. Furthermore, the anchor actuator 41 may again be operated.

In FIGS. 19A and 19B, the extracted nucleic acids and the elution liquidare sent to the analysis chamber 8′ by exploiting the suction pressuregenerated by the pump 25 and the force of gravity. In this step, thefirst and second valve holes 90, 91 are closed by the correspondingshutters 140, 141, and the third valve hole 92 is opened by causingdeformation of the third shutter 142. The pump 25 is active and thegenerated suction pressure causes suction of the liquid on the bottom ofthe extraction chamber 6 and of the extracted nucleic acids (no longerattached to the magnetic beads), through the output fluidic recess 88,the product recess 99′, and the fourth communication hole 100′ (arrow207). In this step, the magnetic beads are withheld in the extractionchamber 6 by the blocking actuator 43 (FIG. 12 ). It is noted that, inthis step, closure of the second valve hole 91 prevents discharge of theextracted nucleic acids into the discharge chamber 7′. The suctionpressure generated by the pump 25 also causes suction and discharge ofthe air in the analysis chamber 8′ towards the fluidic outlet 12 of thecartridge 2′ through the fifth communication hole 102′, the second ventrecess 101′, the third valve hole 92, the fourth vent recess 114′, thethird through hole 113′, the third vent recess 110′, and the secondblind hole 172 (arrow 208).

In this step, the fluidic inlet 11 is connected to the externalenvironment and allows air to flow into the extraction chamber 6 asdescribed above with reference to the arrow 204. Consequently, in FIGS.19A and 19B, the flow of air from outside towards the extraction chamber6 is once again indicated by arrow 204. The air introduced from thefluidic inlet 11 thus rises towards the top of the extraction chamber 6,facilitating displacement of the liquid on the bottom of the extractionchamber 6 towards the analysis chamber 8′.

After transferring the nucleic acids into the analysis chamber 8′, theamplification of the nucleic acids and their analysis is carried out ina per se known manner.

As an alternative to what shown and described, instead of having gaskets120 in the cavities 105, 107 (FIG. 4 ) or in the blind holes 170, 172(FIG. 14 ), the fluidic connection to the connectors 30A, 30B may besimilar to the connection of printer cartridges to the printer. Inparticular, on the cartridge side 2, 2′ a self-closing connectionelement formed by a spring may press a plug on a rubber part functioningas gasket. On the instrument side, just a small cannula may be present,of dimensions compatible with the rubber part, which, during connection,presses the plug, thus compressing the spring.

In the cartridges 2 and 2′, the shape and exact arrangement of thefluidic channels, the holes, and the communication openings may vary.For instance, in the cartridge 2 of FIGS. 4-6 , they may be formed onlyin the body 80, and the first closing wall 81 may be simply a smoothplate similar to the cartridge 2′ of FIGS. 13-15 .

The position of the channels and recesses on the first main face and/oron the second main face 80A, 80B, 80A′, 80B′ of the body 80, 80′ mayvary and comprise a number of stretches formed either on the first mainface 80A, 80A′, or on the second main face 80B, 80B′ of the body 80, 80′or on both of the main faces 80A, 80B, 80A′, 80B′.

In the cartridge 2′ of FIGS. 11-19B, the reagent holes 167 are initiallyclosed and are opened only when the respective containers 165 areconnected to the extraction chamber 6, without any need to be closedagain subsequently. The same applies to the third valve hole 92 (in bothof the embodiments), which is closed until the extracted nucleic acidsare transferred into the collector 8, 8′.

The above holes may then be closed using one-shot valves, in aninexpensive and simple way. In particular, according to one aspect ofthe present description, the one-shot valves are of a material such asto be solid at room temperature and to dissolve when heated, for exampleusing LEDs.

Described hereinafter are possible implementations of one-shot valvesthat may be used in a fluidic circuit for sample preparation cartridges.

For use in LOC devices, it is desirable for the one-shot valves to beinexpensive but reliable, also over time, hermetically separating twoparts of a duct or of a hole. Furthermore, they have to be made ofmaterials that are compatible with the samples and the used reagents andshould not contaminate the liquids.

FIGS. 20-25 show various embodiments of a one-shot valve 209, which mayadvantageously be used in a LOC device, for example in the cartridge 2and 2′ of FIGS. 1-19 , and has the desired characteristics referred toabove. For instance, the one-shot valve 209 may be used as the one-shotvalves 168 of the cartridge 2 (shown in FIG. 13 ) and/or the third valve22 of both of the cartridges 2 and 2′ (FIGS. 5 and 13 ).

According to FIG. 20A, the one-shot valve 209 comprises a duct 210 in avalve body 211 and an obstruction mass 212.

In particular, the obstruction mass 212 is of wax or other inertmaterial that is solid at room temperature or in any case at theoperative temperature, but melts easily and in a controlled way at lowtemperature. For instance, instead of wax, paraffin or other solid fatmay be used, such as cocoa butter, or a gel, such as hydrogel ororganogel. Alternatively, the obstruction mass 212 may be formed by apiece of aluminum. In general, the obstruction mass 212 may be of amaterial that melts at temperatures higher than 60° C. and is inert withrespect to the liquids and to the chemical reactions in the valve body211. The materials referred to above, in particular wax, are suitable tothis end, since they are inert with respect to many chemical reactions,such as those envisaged for LOC application, and thus the contactbetween the obstruction mass 212 and the liquid in the valve body 211does not cause any contamination.

The obstruction mass 212, in the solid state shown in FIG. 20A, has anouter shape corresponding to that of the duct 210, and dimensions suchas to obstruct the latter completely, at a section or lumen 210A thereof(also referred to hereinafter as obstruction section). The obstructionmass 212 thus separates, in the solid state, an upstream portion 210B ofthe duct 210 from a downstream portion 210C and forms a plug.

It is noted that the obstruction section 210A of the duct 210 may alsobe a hole extending through a wall and communicating two portions ofduct that extend on opposite sides of the wall, as, for example, in thecase of the cartridge 2, 2′ of FIGS. 1-19 .

In use, in order to open the one-shot valve 209, the obstruction mass212 is heated and melted until it becomes liquid. In this way, itundergoes deformation and at least partially frees the previouslyobstructed obstruction section 210A (see FIG. 20B, where the obstructionmass is melted and is referred to hereinafter as melted mass 212′). Tothis end, a radiating source 213, for example a LED or a laser, inparticular of an integrated type, is advantageously provided on theinside or on the outside of the valve body 211. When the obstructionmass 212 is of aluminum, the radiating source may be a laser sourcecapable of forming a hole enabling passage of the liquid.Advantageously, the radiating source 213 is focused on the obstructionmass 212 to provide the desired melting energy.

In this way, the obstruction mass 212, which in FIG. 20A obstructs theduct 210, blocking a liquid (designated as a whole by 214) in theupstream portion 210B of the duct 210, in the molten condition (FIG.20B) frees at least in part the section 210A. The liquid 214 may thuspass into the downstream portion 210C, according to the arrow 215. Tothis end, the liquid 214 is subject to a force acting in the directionof arrow 215. For instance, a suction pressure generated by a suctionpump (for example, the pump 25 of FIGS. 1, 2, 11, and 12 ) may beapplied to the downstream portion 210C of the duct, or a pressure actingin the direction of the arrow 215 (not shown) may be applied to theupstream portion 210B; alternatively, another force may be used, forexample the force of gravity or capillarity, with appropriatearrangement, in use, of the duct 210 in the valve body 211.

FIGS. 21A and 21B show an implementation of the one-shot valve 209,wherein, in order to limit the contact between the melted mass 212′ andthe liquid 214, the duct 210 has a collection recess 216, extendingtransversely to the duct 210 in the obstruction section 210A. In thiscase, the obstruction mass 212 (in the non-molten state of FIG. 21A) hasdimensions such as, and is arranged so as, to close the inlet area ofthe collection recess 216. In particular, it has a length, in thelongitudinal direction of the duct 210, and a width, in the directionperpendicular to the drawing sheet, larger than the correspondingdimensions of the collection recess 216. The collection recess 216 maybe a blind hole, arranged at a lower height (in the use position of thevalve body 211) than the obstruction mass 212, or by a peripheralcavity, extending over part or all of the periphery of the obstructionsection 210A of the duct 210, including a bottom area.

When, in use, a melting energy is applied to the one-shot valve 209 andthe obstruction mass 212 melts and becomes fluid, it may penetrate intothe recess 210 by the force of gravity or by capillarity, as shown inFIG. 21B, freeing completely or to a large extent the obstructionsection 210A and opening the one-shot valve 209.

In this case, for example by applying, at appropriate instants, theforce 215, movement of the liquid 214 may be controlled so as to occuronly after the melted mass 212′ has completely gathered in thecollection recess 216 and has re-solidified, thus reducing to a minimumcontact with the liquid 214.

Collection of the melted mass 212′ within the collection recess 216 maybe favored if the obstruction mass 212 contains magnetically sensitivematerial, for example a ferromagnetic material, such as iron filings,and by applying a magnetic field from outside. In this case, movement ofthe melted mass 212′ away from the obstruction section 210A may becontrolled from outside through a magnetic actuator.

For instance, FIG. 22 shows a variant wherein the duct 210 has adischarge branch 217 that branches off from the duct 210, downstream orupstream of the obstruction section 210A, and here extends upwardly (inthe use position of the valve body 211). Alternatively, the dischargebranch 217 may be arranged, in use, at a lower height than thedownstream section 210C of the duct 210. The discharge branch 217 may beclosed, as the collection recess 216 of FIGS. 21A, 21B.

A magnetic actuator 220, external to the one-shot valve 209, shownschematically in FIG. 22 , may be activated simultaneously orimmediately after application of radiant energy to the obstruction mass212. As soon as the latter melts and forms the melted mass 212′, nolonger obstructing the obstruction section 210A, the magnetic actuator220, which attracts the ferromagnetic beads in the melted mass 212′, maybe displaced along the discharge branch 217 (arrow 218), causingdisplacement of the melted mass 212′ inside and along the dischargebranch 217 as far as the tank (not shown). Instead, the liquid 214 isdrawn along the duct 210 as a result of the suction pressure (arrow215). In this way, thanks also to the force of gravity, separation ofthe liquid in the duct 210 and of the melted mass 212′ is ensured.

FIGS. 23A and 23B show an embodiment wherein the valve body 211 istransparent, and a measuring device 219 is arranged along the channel210 in the obstruction section 210A, on the side opposite to theradiating source 213.

For instance, the measuring device 219 may be a photodetector element,such as a photodetector transistor or diode, and an associated circuit,which that measures the amount of current flowing in the photodetectorelement, in a per se known manner.

In this case, when the obstruction mass 212, which is not transparent,is arranged in the obstruction section 210A (FIG. 23A), the measuringdevice 219 detects only the environmental brightness (in this step, theradiating source 213 is also off). After activation of the radiatingsource 213, the measuring device 219 measures in any case a low amountof light, since the majority of light emitted by the radiating source213 is intercepted by the obstruction mass 212, which is opaque. Onlyafter the obstruction mass 212 has melt and the obstruction section 210Ais freed, the light emitted by the radiating source 213 can be detectedby the measuring device 219, which detects a maximum brightnesscondition, corresponding to the valve-open condition.

In this way, it is possible to monitor proper operation of the valve 209both in the closed state (obstruction mass in the obstruction section210A) and in the open state (melted mass 212′ not occupying theobstruction section 210A).

In the embodiment of FIGS. 23A and 23B, the obstruction mass 212 maycontain dark colorants that increase light absorption and speed upmelting. In this way, both a greater brightness difference between theopen and the closed conditions of the valve 209 and a faster actuationare obtained.

The valve 209 of FIGS. 20-25 may be applied, as has been mentioned, to acartridge 2, 2′ of the type described in FIGS. 1-19 . As mentioned, inthis case, the valves 209 shown and described with reference to FIGS.20-25 form the one-shot valves 168 and/or the third valve 22; the valvebody 211 is formed by the body 80, 80′ of the cartridge 2, 2; theradiating source 213 is formed by the melting elements 161 (FIG. 12 );the obstruction section 210A forms the reagent holes 167 or the thirdvalve hole 92; the upstream section 210B is formed by the reagentchambers 165 or by the fourth vent recess 114, 114′ (FIGS. 6 and 15 ,respectively); and the downstream section 210C is formed by the inletfluidic recess 86 or by the second vent recess 101, 101′ (FIGS. 5 and 14, respectively). This solution is shown in FIGS. 24 and 25 , which showthe arrangement of the melting elements 161, 161′ on the turrets 160 and160′, respectively, for a plurality of melting elements 161 (FIG. 24 )or a single melting element 161′, which is vertically mobile so as toface, each time, the one-shot valve 168 to be actuated (FIG. 25 ).

Hereinafter possible implementations of microfluidic connectors that maybe used in sample analysis cartridges are described.

The sample preparation and analysis cartridges are disposable units and,for their use, are connected to machines that generally contain there-usable parts of the preparation and analysis system, includingactuators and control equipment. Connection between a cartridge and thecorresponding machine, enabling exchange of liquids and pneumaticfluids, is obtained through connectors that have the aim of enablingpassage of the fluids hermetically with respect to the externalenvironment.

For microfluidic application, it is thus desirable to have amicrofluidic connector, usable for connecting a cartridge and a controlmachine that is simple to use, safe, fluid-tight, and may bemanufactured using large-scale and low-cost industrial processes.Frequently, it is desirable for the connector to be able to ensurefluid-tightness of at least one of the two parts, even after detachment.

FIGS. 26-30 show embodiments of a connector group 221 that mayadvantageously be used in a LOC device, for example in the cartridges 2and 2′ of FIGS. 1-19 , and that has the above desired characteristics.

In detail, according to FIG. 26 , the connector group 221 comprises amale connector 222 and a female connector 223. They may both be eitherof a disposable or of a non-disposable type, even though generally thefemale connector 223 is of a disposable type.

In the considered example, the male connector 222 comprises a support225, for example of plastic or steel, and a needle 226, generally ofsteel.

The support 225 may have any shape, according to the application.Typically, it is formed by a hollow cup-shaped body, having firstmounting means 225A (for example, external screw means), for attachingit to a fixed supporting structure 227, and second mounting means 225B,bonded, for example welded, to a fluidic line 228. For instance, in thecase of the machine 3 or 3′ of FIGS. 1 and 11 , the support 225 may be acup-shaped plastic body, screwed on the plate 51 of the machine 3, 3′(FIG. 2 ) and glued or welded to a tubular duct portion that forms or isconnected to the ventilation line 33, to the first fluidic duct 54,and/or to the pneumatic duct 55 (which form the fluidic line 228).

The needle 226, which has a generally cylindrical shape, is similar tohypodermic needles and thus has a smooth lateral surface, with verylimited roughness so that it is unlikely to trap harmful agents.Furthermore, the needle 226 has a supporting end 226A, bonded, forexample welded or glued, to the support 225, and a tip end 226B, whichis sharp and pointed. The needle 226 is hollow and has an injectionchannel 230 longitudinally extending from the supporting end 226A up toa lateral opening 230A. The injection channel 230 thus openslongitudinally with respect to the needle 226 towards the inside of thesupport 225 and is in fluidic connection, through the support 225, withthe fluidic line 228. The lateral opening 230A of the injection channel230 is arranged alongside the needle 226 near the tip 226A. Thus, thetip 226A of the needle 226 is closed and not perforated.

The fluidic line 228 is generally connected to a fluid actuator (notshown), which can be operated manually or automatically, such as apiston mobile in the support 225 or an external pump that generates apositive or negative pressure within the support 225, or some otheractuator.

The female connector 223 comprises a containment body 235 forming aconnector chamber 236 housing a gasket 240. The female connector 223 maybe of plastic, for example as discussed below with reference to FIG. 28.

In the shown embodiment, the containment body 235 has a generallyparallelepipedal outer shape with two opposite lateral faces, designatedby 235A and 235B in FIG. 26 , which have, respectively, a needle-entryhole 241 and a fluid opening 242, typically not aligned with each other.The outer shape of the containment body 235 and the position of theneedle-entry hole 241 and of the fluid opening 242 may, however, vary,according to the application; for example, the containment body 235 mayform part of a more complex structure, for instance of the cartridge 2or 2′ of FIGS. 4-6 and 13-15 , as discussed hereinafter.

The needle-entry hole 241 extends between the lateral face 235A of thecontainment body 235 and the connector chamber 236 and is shaped tofacilitate introduction of the needle 226. Instead, the fluid opening242 is connected to the connector chamber 236 with a duct 244, hereL-shaped, which opens onto the connector chamber 236 at a duct opening244A. In particular, the duct opening 244A is formed on a face of theconnector chamber 236 (duct face 236A) adjacent to the face — needleface 236B — where the needle-entry hole 241 opens. Thus, the duct face236A is not opposite to the needle face 236B, for the reasons explainedhereinafter.

The gasket 240 is cup-shaped with rectangular base and rounded edges(FIG. 27 ) and comprises a sidewall 243A and a bottom wall 243B, whichdelimit a gasket cavity 245. The gasket cavity 245 is faces the ductface 236A of the connector chamber 236 and thus opens towards the ductopening 244A, whereas its bottom wall 243B rests against the face of theconnector chamber 236 opposite to the duct face 236A (the bottom face236C). In some applications, the bottom wall 243B may be missing.

According to an embodiment, the surface of the gasket 240 facing theduct face 236A of the connector chamber 236 has a projecting profile orstep 246. The projecting profile 246 surrounds the gasket cavity 245 andbears upon the duct face 236A of the connector chamber 236. The rest ofthe surface of the gasket 240 facing the duct face 236A of the connectorchamber 236 thus forms a peripheral lowered portion 247 surrounding theprojecting profile 246. In this way, the gasket 240 does not rest withits entire top surface against the duct face 236A of the connectorchamber 236, thereby increasing the pressure exerted by the gasket 240on the duct face 236A (for a same force), and thus has an excellenttightness even in case of not perfectly flat surfaces of the containmentbody 235 or of the projecting profile 246 (for example, having a certaindegree of roughness).

The material of the gasket 240 is typically rubber, for example siliconerubber, thereby the gasket 240 has a high elasticity, may be easilyperforated by the needle 226, has a hardness such as to withstandmultiple needle insertion and extraction cycles, has a good seal aroundthe hole where the needle is introduced, and is chemically inert withrespect to the substances injected or drawn off. Typically, the materialof the gasket 240 has a value on the Shore-A scale comprised between 15and 45 Shore A, for example 20 Shore A. Other materials suitable for thegasket 240 are, for example, fluorosilicone (with a hardness of between30 and 80 Shore A) and neoprene (with a hardness of between 20 and 90Shore A).

The female connector 223 may be manufactured by injection co-moldingenabling molding of plastic (to form the containment body 235) andrubber (to form the gasket 240), using various channels for injectioninto a same mold, or a multiphase injection process, or by any othermolding method known in the art, so as to form the gasket 240 directlyin the containment body 235.

Alternatively, the containment body 235 may be made of two distinctparts, bonded together after insertion of the gasket 240, as shown inFIG. 28 . Here, the containment body 235 is formed by a housing portion250 and a lid 251. The housing portion 250 is, for example, of moldedplastic material and houses the connector chamber 236, the duct portion244 and the needle-entry hole 241. The lid 251 is, for example, formedby a plane plate, also of plastic material. The housing portion 250 andthe lid 251 are bonded together using any suitable technique, forexample hot rolling or a thermal and/or pressure process.

With the embodiment of FIG. 28 , prior to assembling the femaleconnector 223, the projecting profile 246 of the rubber gasket 240 mayhave a slightly greater height than the connector chamber 236. Inparticular, as shown in FIG. 28 , the connector chamber 236 may have aslightly lower depth dh than the maximum height dr1 of the gasket 240 atthe projecting profile 246. In this case, the height dr2 of the gasket240 at the lowered portion 247 may be slightly lower than the depth dhof the connector chamber 236.

For instance, for a connector chamber 236 having a volume of 120-130 μl,and the gasket cavity 245 having a volume of 10-20 μl, dh may be 2.95mm, dr1 may be 3 mm, and dr2 may be 2.9 mm.

For the embodiment of FIG. 28 , the female connector 223 is assembled byintroducing the gasket 240 into the connector chamber 236, with the topsurface of the gasket 240 (formed by the projecting profile 246 and thelowered portion 247) against the duct face 236A of the connector chamber236, and thus with the gasket cavity 245 facing the duct face 236A. Inaddition, part of the sidewall 243A of the gasket 240 arranges adjacentand contiguous to the needle face 236B of the connector chamber 236 toclose the needle-entry hole 241. Then, the lid 251 is bonded to thecontainment body 235. In this step, the projecting profile 246 of thegasket 240 is slightly compressed, thus ensuring perfect adherence ofthe gasket 240 to the duct face 236A of the connector chamber 236 andinterference seal. The presence of the lowered portion 247 enablespossible deformation of the projecting profile 246 and lateral wideningthereof, if desired.

In use (FIG. 29 ), the needle 226 of the male connector 222 isintroduced into the needle-entry hole 241, which functions as insertionguide, and perforates the sidewall 243A of the gasket 240 until itpenetrates into the gasket cavity 245. Then a fluid, either a liquid ora gas, may be injected by the needle 226 towards the duct portion 244(as indicated by the arrow 248) or, vice versa, drawn from the fluidopening 242, through the duct portion 244, the gasket cavity 245, andthe needle 226, as far as the support 225.

The connector group 221 is thus shaped to ensure a perfect seal duringthe steps of suction/injection of a fluid. In fact, the overlaying ofthe materials (harder material for the containment body 235 on the ductface 236A of the connector chamber 236, softer material for the gasket240, and harder material for the containment body 235 on the bottom ofthe connector chamber 236) ensures hermetic sealing of the gasket 240 ina lasting way. In particular, the projecting profile 246 causes amechanical compressive stress in the area (around the duct opening 244Aof the duct 244) where hermetic sealing is required, without requiring aperfect adhesion over the entire surface of the gasket 240, which ismore difficult to guarantee in a perfect way in each point of the entiresurface of the connector chamber 236, in case of intrinsic defectivenessof the material, such as surface roughness.

Furthermore, the arrangement of the needle-entry hole 241 on the needleface 236B, adjacent, and not opposite, to the duct face 236A of theconnector chamber 236 (at the duct opening 244A) contributes totightness of the connector group 221 during injection and suction. Itmoreover facilitates manufacture of the female connector 223, in thecase of production in two pieces since the gasket cavity 245 is simplyclosed by bonding the lid 251 to the containment body 235 (FIG. 28 ).

During introduction of the needle 226, thanks to the closed shape of itstip 226B and the transverse arrangement of its lateral opening 230A, theneedle 226 does not cause detachment of any portion of the gasket 240(thus preventing the risk of core drilling) and thus does not createswarf that might enter the needle 226 or penetrate into the fluidiccircuit connected to the connector group 221 and block the fluid flow.

With the solution shown in FIGS. 24-27 , the female connector 223 isintrinsically sealed since the rubber of the gasket 240 is self-sealingand thus ensures tightness even when the female connector 223 isseparated from the male connector 222 (when the needle 226 isextracted). When it is desired to ensure tightness on the male connector222 after extraction of the needle 226, it is possible to arrange amicrofluidic valve of any type upstream of the needle 226, asrepresented with a dashed line and designated by 254 in FIG. 29 .

As referred to above, the connector group 221 may be used in the system1 and 1′ according to FIGS. 1-19 . In particular, the connector group221 may form the connection element 30A and the fluidic inlet 11 or theconnection element 30B and the fluidic outlet 12, with the correspondinggaskets 120, of the system 1 or 1′ of FIGS. 1 and 11 .

For instance, FIG. 30 shows the cartridge 2′ used in the system of FIG.11 and shown in detail in FIGS. 13-15 . In this case, the containmentbody 235 is formed by the cartridge 2′, the connector chamber 236 isformed by one of the blind holes 170, 172, the gasket 240 forms thegasket 120 of FIG. 13 , the duct 244 forms the inlet fluidic recess 86or the third vent recess 110′, and the duct opening 244A of the duct 244forms the inlet hole 171 or the first communication hole 175.Furthermore, the support 225 may be formed by the plate 51 (FIG. 2 ),and the needle 226 may form one of the needles 58 of the connectionelement 30A or 30B. Furthermore, the housing portion 250 (FIG. 28 ) maybe formed by the body 80′ of the cartridge 2′, and the lid 251 may beformed by the first closing wall 81 (FIG. 13 ).

Likewise, for the cartridge 2 of FIGS. 4-6 , the connector chamber 236forms the chamber-like recesses 105, 107, the needle-entry hole 241forms the cavities 104, 106, and the duct opening 244A of the duct 244forms the through holes 87, 108.

Hereinafter, possible implementations of containers for gatheringsamples, also defined as test tubes, are described.

Microfluidic devices of a LOC type, to be able to carry out sampleanalysis, have an inlet for introducing a sample to be treated andanalyzed. In these devices, it is desirable that loading is safe andavoids any possibility of cross-contamination, i.e., any type ofcontamination of the sample by the operator performing the loading andany type of contamination of the operator by the material of the sample.It is moreover desirable that loading is simple and does not requireparticular skills or attention by the operator to enable execution of awide range of analyses, without any need for skilled persons.

To this end, it is advantageous to use a container that is easy toattach to the microfluidic device, enables easy introduction of samples,is portable, prevents any contamination, enables introduction of smallamounts of liquids to be analyzed, entailing minimum invasiveness forthe patient, and has a low cost.

FIGS. 31-37 show various embodiments of containers 260 that mayadvantageously be used in a LOC device, for example in the cartridge 2,2′ of FIGS. 1-19 , and that have the desired characteristics referred toabove. For instance, the container 260 may co-operate with the sampleinlet 10 of FIG. 4 or FIG. 9 , as explained in detail hereinafter.

According to FIG. 31 , the container 260 comprises a tubular body 262and a lid 263 and is designed to be attached to a container support 261.

The tubular body 262 is typically of plastic, for example polyethyleneterephthalate, and is substantially vial-shaped, with a tubular wall262A, a bottom end 262B having a tapered shape, and an open top end262C. Here, the terms “bottom” and “top” refer to the use position ofthe container 260.

The bottom end 262B is closed by a perforable wall 262D, which may be asingle piece with the tubular wall 262A and is configured to be easilyperforated. In this case, the perforable wall 262D is of the samematerial as the tubular wall 262A, but thinner. For instance, typicalthicknesses of the tubular wall 262A and of the perforable wall 262Dare, respectively, 1 mm and from 0.1-0.3 mm. Alternatively, theperforable wall 262D may be of a softer material than the tubular wall260A, for example rubber.

The tubular body 262 has a blocking structure here formed by a firstseal ring 264 slid on the tubular wall 262A. The first seal ring 264 isof elastomeric material, for example Viton, and may co-operate with acorresponding stop 265 on the container support 261, as explained below.In this case, the blocking structure 264 also functions as a sealingstructure and prevents, in the event of leakage during or afterperforation of the perforable wall 262D, part of the analyzed samplefrom possibly escaping into the external environment.

Alternatively, the blocking structure may be made in any other way, forexample as peripheral projection that hooks onto a correspondingattachment portion on the container support 261 or that snaps into acavity or behind a projection on the container support 261.

Moreover, the tubular body 262 has a guide structure 266, for properinsertion of the container 260 into the container support 261. Here, theguide structure 266 of the container 260 is formed by a peripheralribbing (which, for reasons of simplicity, is again designated by 266)that projects from the tubular wall 262A near the bottom end 262B, andthus lower down (in the use position) than the first seal ring 264. Theperipheral ribbing 266 may be just one and extend over all or over amajor part of the circumference of the tubular wall 262A, or be formedby a plurality of portions (at least two) arranged radially at adistance from each other.

The peripheral ribbing 266 has a certain elasticity to be able toundergo deformation and overcome the stop 265 on the container support261 (as explained below). The ribbing may have a triangular ortrapezoidal cross-section, with a bottom surface 266A (closer to thebottom end 262B) with oblique orientation in order to facilitateintroduction thereof into the container support 261, and a top surface266B (facing the top end 262C of the tubular wall 262) that issubstantially perpendicular to the tubular wall 262 in order to blockthe tubular body 262 vertically in the container support 261 afterinsertion, as explained in greater detail hereinafter. Alternatively,the peripheral ribbing 266 may be relatively stiff, and the stop 265 maybe more elastic. According to another possibility still, both theperipheral ribbing 266 and the stop 265 may be elastic.

The tubular body 262 further has lid attachment means 263, here formedby a thread 269 external to the tubular wall 262A and arranged near thetop end 262C. The top end 262C of the tubular body 262 also has alid-guide structure 270, here formed by a guide tooth, which extends inthe tubular wall 262A. The guide tooth 270 may be just one and have acircumferential extension, or be formed by a number of parts, as isclear to the person skilled in the art.

The lid 263, which is typically of plastic material, for example thesame plastic material as the tubular body 262 (polyethyleneterephthalate), comprises a base portion 263A, a screwing portion 263B,and a plug portion 263C. In detail, the base portion 263A has a flatcylindrical shape and is typically designed to close the top end 262C ofthe tubular body 262, the screwing portion 263B has the shape of acylindrical wall projecting peripherally from the base portion 263A, andthe plug portion 263C extends centrally from the base portion 263A onthe same side as the screwing portion 263B. The screwing portion 263Binternally has a structure for fixing to the tubular body, here acounter-thread 271, designed, in use, to be screwed on the thread 269 ofthe tubular body 262. Thus, the screwing portion 263 has an internaldiameter slightly greater than the external diameter of the tubular wall262A and, in the closed condition of the container 260, extends outsidethe tubular wall 262A. The plug portion 263C has a cylindrical shape,here full, with a slightly smaller diameter than an internal diameter ofthe tubular wall 262A so as enable it to be fitted in the top end 263Cof the tubular body 262 when the lid 263 is screwed thereon.

In addition, the plug portion 263C has a greater height (in thelongitudinal direction of the tubular body 262) than the screwingportion 263B, for the reasons explained below. Furthermore, the plugportion 263 carries a second seal ring 272 slid on the plug portion 263that has an external diameter substantially equal to or slightly greaterthan the internal diameter of the tubular wall 262A so as to sealhermetically the inside of the tubular body 262.

The container support 261 is designed to receive the bottom end 263B ofthe tubular body 262 and fluidically connect the inside of the container260 to a fluidic circuit, as shown in FIGS. 32A and 32B.

The container support 260 is here formed by a cylindrical wall 275 witha circular base extending from a connection portion 277 fixed to, forexample integral to, a LOC device, such as the cartridge 2 or 2′ ofFIGS. 4-6 and 13-15 , at the sample inlet 10, and has acontainer-introduction end 275A. The container support 261 may be asingle piece with the connection portion 277 or may be manufacturedseparately and bonded, for example glued or fluidically connected by anytype of stable sealed connection. Near the connection portion 277, thecontainer support 261 has an own guide structure 276 intended to couplewith the guide structure of the container 260. In the consideredexample, the guide structure 276 of the container support 261 is formedby an internal thread (which, for reasons of simplicity, is once againdesignated by 276), intended to engage the peripheral ribbing 266 of thecontainer 260. Furthermore, near its container-introduction end 275A,the cylindrical wall 275 of the container support 260 has the stop 265referred to above that is designed to co-operate with the first sealring 264, for blocking the container 260 in use (FIGS. 32A and 32B). Thestop 265 may comprise, for example, an internal peripheral projection,formed near the container-introduction end 275A of the cylindrical wall275. The stop 265 advantageously has an inclined top surface 265A tofacilitate passage of the peripheral ribbing 266 of the tubular body 262and of the first seal ring 264 during introduction of the container 260,and a bottom surface 265B, perpendicular to the cylindrical wall 275, toprevent exit of the first seal ring 264 after it has overcome the stop265. In this step, as mentioned, the peripheral ribbing 266 of thetubular body 262 and the first seal ring 264 undergo elasticdeformation, to overcome the stop 265.

As an alternative to what shown, the guide structure 276 of thecontainer support 261 may be arranged near the container-introductionend 275A, and the stop 265 may be arranged between the guide structure276 of the container support 261 and the connection portion 277.

The connection portion 277 has, at the center of the container support261, a perforation structure 278 projecting towards the inside of thecylindrical wall 275 of the container support 261. The perforationstructure 278 is hollow, has a pointed shape, and is in fluidicconnection with a fluid-communication line 279 formed in the connectionportion 277.

In use, prior to bonding the container 260 to the container support 261,the tubular body 262 is filled with a sample to be analyzed. Filling maybe carried out in different ways: for example using an external pipetteor as described below with reference to FIGS. 33-35 . When it is filledusing a pipette (not shown), after filling, the lid 263 is screwed onthe tubular body 262. In this case, since, during screwing, the plugportion 263C fits and extends into the tubular wall 262A, it causes anoverpressure within the container 260, which facilitates subsequenttransfer of liquid. In this step, the second seal ring 272 seals thesample in the container 260 (FIG. 32A) from the external environment.

Then, the container 260 is inserted and screwed into the containersupport 261, here through the engagement of the peripheral ribbing 266of the tubular body 262 with the internal thread 276 of the containersupport 261. Screwing ensures a correct guide and exact positioning ofthe container 260, and in particular of the perforable wall 262D, which,during screwing, is thus easily perforated by the perforation structure278 (FIG. 23B). At the end of insertion of the container 260, the firstseal ring 264 blocks the container 260 in position inside the containersupport 261, preventing extraction thereof, and ensures that the sampledoes not spill out, as explained above.

The liquid in the container 260 may thus flow into thefluid-communication line 279, as indicated by arrow 280, thanks to theoverpressure generated by the plug portion 263C, as explained above, andpossibly aided by gravity.

FIG. 33 shows a container 260 enabling a different method for loadingthe sample, which may advantageously be used for sampling blood from apatient. Here, the lid 263 is connected to a catheter 283, or othercannula or tube. In particular, the catheter 283 has a first end 283Aconnected, for example, to a sampling needle 284, possibly through avalve needle 285 (for example, of a type common for blood sampling), anda second end 283B extending through the base portion 263A and the plugportion 263C of the lid 263 and in fluidic connection with the inside ofthe container 260.

In this way, once the sampling needle 284 has been positioned in a bloodvessel of a patient and the valve needle 285 has been opened, thedifference in pressure between the inside of the container 260 and theblood vessel draws in the blood. Next, after closing the valve needle285, the container 260 is fitted into the container support 261,perforating the perforable wall 262D and enabling the blood to flow inthe fluid-communication line 279, as has been described above withreference to FIGS. 32A and 32B. Alternatively, the container 260 may beinserted into the container support 261 prior to sampling.

With the solution of FIG. 33 , it is advantageous that the container 260does not require opening of the lid 263 to load the sample, to theadvantage of sterility of the system.

FIG. 34 shows another way for loading the container 260. Here, the lid263 is not monolithic, but the plug portion 263C has a core 287 of aperforable and re-closable material, such as rubber, in particularsilicone rubber. The core 287 extends throughout the height of the plugportion 263B and through the base portion 263A.

The core 287 can thus be easily perforated by a filling needle 288 forinjecting the sample into the container 260. Then, the filling needle288 is extracted. However, the elastic material of the core 287 ensuresreclosing of the injection hole, keeping the inside of the container 260sealed from the external environment.

The container 260 of FIG. 35 enables sampling and transferring a samplealso of a solid type. Here, the plug portion 263C of the lid 263 carriesa rod 290 ending with a swab 291 of a type commonly used for samplingsolid material. Moreover, an elution liquid 293 is present within thecontainer 260.

In this case, after sampling the solid material with the swab 291, thelid 263 is screwed on the tubular body 262, causing immersion of thetaken solid sample in the elution liquid 293. The solid material, thusdissolved in the elution liquid 293, may be transported by the elutionliquid 293 to the fluid-communication line 279 (FIG. 32 ) afterperforation of the perforable wall 262D.

FIG. 36 shows the container 260 of FIG. 31 applied to the cartridge 2 or2′ of FIG. 5-7 or 13-15 . Here, the cylindrical wall 275 of thecontainer support 261 projects from the top face 80C, 80C′ of the body80, 80′, as the perforation structure 278. In addition, the cylindricalwall 275 and the perforation structure 278 are one piece with the body80, 80′. Here, the inside of the perforation structure 278 is directlyconnected to the extraction recess 83 through the introduction opening117, closed by the plug element 89. In a not shown manner, thecylindrical wall 275 of the container support 261 may be equipped withthe guide structure 276 of the container support 261, as for thecontainer 260 of FIG. 31 .

In the embodiment of FIG. 37 , the container support 261 has a supportplug 295, of an incorporated type, shown with a solid line in a closedposition and with a dashed line in the open position. The support plug295, which may be of the type used in test tubes, is here one piece withthe cylindrical wall 275 of the container support 261. Alternatively,the support plug 295 may be slid on or otherwise coupled to thecylindrical wall 275 of the container support 261. The support plug 295comprises a stem 296, which extends from the cylindrical wall 275, and acap 297. The stem 296 is flexible and forms a hinge that enables openingand closing of the incorporated plug by simply flipping over the cap297, as shown in FIG. 37 with a solid line (closed position) and with adashed line (open position). The cap 297 has a closing portion 297A,which, in the closed position of the incorporated plug 295, extendstransversely to the cylindrical wall 275 of the container support 261,closing the container-introduction end 275A of the latter, and anengagement portion 297B, projecting from the closing portion 297A andwith a cylindrical shape, designed to be inserted into the cylindricalwall 275 of the container support 261 and to engage with interferencefit the cylindrical wall 275 of the container support 261, for exampleat the stop 265 or internal thread 276 of the cylindrical part 275, byexploiting the elasticity of the material. To this end, the engagementportion 297B of the cap 297 has an external diameter equal to orslightly greater than, an internal diameter of the cylindrical wall 275at the stop 265 or than the internal thread 276, to be blocked by one ofthese, in a closed position of the support plug 295.

The incorporated plug 295 may thus be easily manually opened whileinserting the container 260.

In the case of application to the cartridge 2 or 2′, since the container260 has no air inlet to compensate for the outlet of liquid, the pump 25(FIGS. 1 and 11 ) is sized so as to enable emptying of the container260. Alternatively, the pump 25 may carry out a sequence of suction andinsufflation steps to facilitate transfer of all the liquid from thecontainer 260 to the inside of the chamber 6 (inside the extractionrecess 83).

According to another embodiment, air compensation exploits the lateralopening 118 of the cartridge 2 (FIGS. 5-7 ). The same process may beapplied for the cartridge 2′ (FIGS. 13-15 ).

Advantageously, the container 260 is readily usable, has reduced costs,is robust, and ensures the desired sterility level. The containersupport 261 may be easily provided on a connection portion 277, such asa microfluidic cartridge.

Possible implementations of magnetically controlled valves are describedhereinafter.

Microfluidic devices comprise fluidic paths integrated in the device andformed by channels, openings, holes, etc., which are opened and closedaccording to the treatment steps. To this end, microvalves may generallybe used that can be controlled from outside.

It is thus desirable for these valves to be simple, inexpensive, andreliable, ensure the possibility of being easily integrated in themicrofluidic device, and be compatible with the liquids treated.

FIGS. 38-42 show various embodiments of a magnetic valve 300 that mayadvantageously be used in a LOC device, for example in the cartridges 2and 2′ of FIGS. 1-19 and has the desired characteristics referred toabove. For instance, the magnetic valve 300 may form the valves 20-22shown in FIGS. 1-19 .

In detail (FIG. 38A), the magnetic valve 300 comprises a valve body 301and a shutter 302 and co-operates with an actuator 303. The magneticvalve 300 and the actuator 303 form a valve group 304.

The valve body 301 forms a fluidic path 305, here comprising a firstpath portion 306 and a second path portion 307. The path portions 306,307 are here arranged transversally, for example perpendicular, withrespect to each other. In particular, here, the second path portion 307ends at the first path portion 306 at an opening 307A, to form aT-coupling 309. Here, the first path portion 306 is a duct, hasrectangular or square section and defines a wall 306A facing the secondpath portion 307. The second path portion 307 may be a duct or a holeleading to another duct, and have a section of any shape, for examplecircular, rectangular, or square.

The wall of the valve body 301 forms, around the opening 307A, aperipheral projection 308 that extends towards the inside of the firstpath portion 306.

The shutter 302 is formed by a magnetically deformable membrane arrangedinside the first path portion 306 at the coupling 309. The shutter 302is thus arranged in front of the opening 307A of the second path portion307 and is configured to close the opening 307A when the shutter 302 isin the undeformed condition and to free at least one part of the opening307A when the shutter is in the deformed condition.

In detail, the shutter 302 is here formed as a single piece ofelastically deformable ferromagnetic material, typically of softbicomponent rubber incorporating ferrite particles or powder, ironfilings, and, in general, powder of materials that are susceptible to amagnetic field. For instance, in case of ferrite, it may be 66% of thetotal weight.

In the embodiment of FIGS. 38A, 39A, and 39B, in the undeformedcondition, the shutter 302 is substantially frustoconical with a majorbase 302A and a minor base 302B; moreover, a cylindrical portion ofsmall height forms the major base 302A. The shutter 302 is moreoverarranged with the major base 302A against, and in contact with, theperipheral projection 308 and with the minor base 302B against, and incontact with, the wall 306A of the first path portion 306. The majorbase 302A of the shutter 302 has a greater diameter than the opening307A, if the latter has a circular shape. In any case, the major base302A of the shutter 302 has a greater area than the opening 307A, and ashape and position such as to cover and completely close the opening307A.

Furthermore, the height H1 of the shutter 302 (height of thefrustoconical portion) is equal to, or slightly greater than, the heightH2 of the first path portion 306 (or the dimension of the first pathportion 306 in the considered section, in a perpendicular direction tothe wall 306A of the first path portion 306). In this way, thanks to theelasticity of the shutter 302, in the undeformed condition, the shutter302 is slightly pressed within the fluidic path 305 and reliably closesthe opening 307A. The fluidic connection between the first and secondpath portions 306, 307 is consequently interrupted, and a fluid, forexample a liquid, 310 in the first or in the second path portion 306,307 (in FIG. 38A, in the first path portion 306) can thus not flow intothe other path portion 307, 306.

Advantageously, in the embodiment of FIGS. 38-39 , where the first pathportion 306 is located upstream and the second path portion 307 islocated downstream of the fluidic path 305, the fluid 310 pushes againstthe conical wall of the shutter 302, and the pressure of the fluid 310favors adhesion of the shutter 302 against the opening 307A. Theadhesion effect is increased when an external force is applied on thefluid 310 in the fluidic path 305 in order to push the fluid 310 towardsthe second path portion 307 or draw it into the second path portion 307.

The actuator 303 is of a magnetic type and generates a magnetic field B,when activated. For instance, the actuator 303 may be formed by a coilelectromagnet activated when it is traversed by a current.Alternatively, the actuator 303 may be formed by a permanent magnetmoved to and away from the valve body 301 for respectively controllingopening and closing of the valve 300. The actuator 303 faces the valvebody 301 in proximity of the wall 306A to be closer to the minor base302B than to the major base 302A of the shutter 302, or in any case isbrought into this position when activated.

When the actuator 303 is activated (turned on or moved to the coupling309 of the valve 300), the thereby generated magnetic field B attractsthe ferrite particles or powder and causes deformation of the shutter302, as shown in FIGS. 38B and 40 . In practice, the conical portion ofthe shutter 302 forms a circular “wing” (on the sides, in FIG. 38B) thatis attracted by the actuator 303 and turns over towards the minor base302B of the shutter 302, detaching from the peripheral projection 308and freeing the opening 307A. The fluid 310 in the first duct portion306 can thus flow towards the second duct portion 307 in the directionindicated by the arrows of FIG. 38B.

When the actuator 303 is deactivated (turned off or moved away), thanksto the elasticity of the material of the shutter 302, it returns intoits undeformed configuration, thus closing again the opening 307A.

With the embodiment of FIGS. 39 and 40 , excellent valve closing andease of actuation are thus obtained. In particular, thanks to itssymmetry, the shutter 302 exerts a uniform sealing action on its entirecontact surface (peripheral ribbing 308) in the closed condition,providing a maximum effectiveness and sealing reliability. Furthermore,when the shutter is deformed by the actuator 303, it undergoesdeformation in a symmetrical way, preventing internal stresses caused bystiffness differences due to geometrical reasons.

Alternatively, the shutter 302 may have a frustopyramidal shape, afrustoprismatic shape, or a more complex shape.

FIGS. 41-43 show shape variants of the shutter 302, designated by 315,318, and 319, respectively, which may be useful in particularconditions, for example in case of a first path portion 306 of a largewidth. The shutters 315, 318 and 319 are formed by two parts: a stemportion 316 and a shutter portion 317, bonded together. The stem portion316 and the shutter portion 317 may be of a different material. Forexample, the stem portion 316 may be non-ferromagnetic and/ornon-elastic material, such as plastic, metal, or a polymer and may thusbe deformable or not. The shutter portion 317 is, instead, offerromagnetic elastic material, as described above for the shutter 302.

In all of FIGS. 41-43 , the base portion 316 of the shutter 315, 318,319 rests against, for example is bonded to, the wall 306A of the firstpath portion 306, and the shutter portion 317 rests against theperipheral ribbing 308 with its major base 317A that closes the opening307A.

In detail, the shutter 315 of FIG. 41 has a frustoconical-shaped shutterportion 317, as in FIGS. 39A and 39B, with its major base 317A incontact with the opening 307A and its minor base 317B having a greaterarea than the base portion 316.

Alternatively, the open/close portion 317 of the shutter 315 may befrustopyramidal-regular prism-shaped.

The shutter 318 of FIG. 42 has a parallelepiped- cube- orcylinder-shaped shutter portion 317, with a greater area than the baseportion 316.

The shutter 319 of FIG. 43 has a cone-, pyramid-, or prismaticring-shaped shutter portion 317, with a central hole through which thebase portion 316 is inserted.

In all the solutions of FIGS. 39-43 , the valve body 301 may be a singlepiece and the shutters 302, 315, 38 and 319 may be pressed into thevalve body 301. Alternatively, the valve body 301 may be made up of twoparts: a first part 301A housing the first and second path portions 306,307 (where the first path portion 306 is open at the side intended toform the wall portion 306A of the first path portion 306); and a secondpart 301B closing the first path portion 306, forming the wall portion306A. For instance, the second part may be a chip or a film. In thisway, insertion of the shutter is facilitated, and the shutter 302, 315,318, and 319 is slightly compressed when the two parts of the valve bodyare bonded together.

As referred to above, the connector group 221 may be used in the system1 and 1′ according to FIGS. 1-19 . In particular, the shutter 302, 315,318, or 319 may form the magnetic bodies 140-142.

For instance, FIG. 44 shows the cartridge 2′ used in the system of FIG.11 and shown in detail in FIGS. 13-15 . In this case, the valve body 301may form the main body 80′, and the second closing wall 82; the shutter302/315/318/319 forms the magnetic bodies 140-142, as referred to above;the first path portion 306 forms the first vent recess 95, the outputfluidic recess 88, and the second vent recess 101; and the second pathportion 307 forms the first, second, and third valve holes 90-92. Thesame applies to the cartridge 2 used in the system of FIG. 1 and shownin detail in FIGS. 4-6 , with the difference that the first path portion306 forms the first vent recess 95, the output fluidic recess 88, andthe second vent recess 101. Thus, in a not shown manner, the valve holes90-92 of the cartridges 2 and 2′ may have a respective peripheralprojection 308 (not shown).

For the cartridges 2 and 2′, closing of the second path portion 307(valve holes 90-92) is favoured by the suction pressure applieddownstream of the valve holes 90-92, as described in detail in FIGS.7-10 for the cartridge 2 and in FIGS. 16-19 for the cartridge 2′.

When incorporated in the cartridge 2 or 2′, the shutter 302 (the shutterportion 317) has a diameter of the major base 302A of 4-7 mm, typicallyabout 6 mm, a diameter of the minor base 302B of 1.5-4 mm, typicallyabout 2.3 mm, and an overall height of 1-mm, typically about 1.3 mm, andthe cylindrical portion (forming the major base) has a height of 0.1-0.3mm, typically about 0.2 mm.

In the shown embodiment, the magnetic valve 300 forms a normally closedvalve, opened by deformation of the shutter or shutter portion 302, 317.It thus enables a duct/hole/channel/recess to be closed in a reliable,simple, and inexpensive way and to be controlled using a simple magneticactuator. With the shown arrangement, with the major base 302B facingthe downstream duct portion (second path portion 307), the fluidpressure and possible forces acting on the fluid favor tightness. Thepresence of the peripheral projection 308 in turn favors hermetic seal,since the compression of the elastic material forming the shutter 302(or the shutter portion 317) generates a concentrated force in a smallarea (area of contact between the shutter 302 or shutter portion 317 andthe peripheral projection 308).

Even though FIGS. 38-44 show a magnetic valve 300 arranged at theT-coupling 309 between two path portions 306, 307, with an appropriategeometry of the coupling portion, the magnetic valve 300 is able toreliably close even two fluidic path portions arranged at an angle otherthan 90° or even aligned.

Possible implementations of a system for stirring and mixing liquids aredescribed hereinafter and may be used in portable microfluidic devices,such as cartridges for analysis of biological samples, to whichreference is made to hereinafter, without any loss of generality.

In cartridges for the analysis of biological samples, due to their smalldimensions and their use outside specialized laboratories and by personswithout particular know-how and skills, the problem exists of enablingthe intended reactions in the chambers for performing analysis ofbiological samples in a reliable way, in short times, and with sureresults.

To this end, it is useful to have solutions that enable effective mixingof the liquids in the reaction chambers, notwithstanding the smalldimensions of the chambers.

FIGS. 45-47 show an embodiment of a stirring and mixing group 320 havingthe desired characteristics referred to above and advantageously usablein a microfluidic device during treatment of a liquid sample. Forinstance, the stirring and mixing group 320 may be used in thecartridges 2 and 2′ of FIGS. 1-19 , as discussed hereinafter.

According to FIG. 45 , the stirring and mixing group 320 comprises amagnetic generator 325 and a microfluidic device (cartridge) 323 havinga reaction chamber 322 accommodating a ferromagnetic anchor 321. Theferromagnetic anchor 321 is of ferromagnetic material; for example, itmay be completely of stainless steel, or plastic-coated iron, or of anyother non-oxidable ferromagnetic material or any other oxidableferromagnetic material coated with non-oxidable material and inert tothe reaction that takes place in the reaction chamber 322.

The ferromagnetic anchor 321 is shaped as a cylindrical rod having alength such as to be able to move inside the reaction chamber 322.

The ferromagnetic anchor 321 is subject to a magnetic field generated bythe magnetic generator 325, arranged outside the microfluidic device323. Typically, the magnetic generator 325 comprises a magnetic element326 configured to generate a rotating magnetic field. Here, the magneticelement 326 is formed by a permanent magnet 324 mounted on a d.c. motor328, which can turn horizontally so as to drive the permanent magnet 324in rotation. Furthermore, the magnetic element 326 of FIG. 45 can betranslated along the reaction chamber 322. In the shown example, thereaction chamber 322 has a larger extension in a vertical direction. Inthis case, the magnetic element 326 is mobile vertically, as indicatedby arrow 329. For instance, the magnetic element 326 may be carried by asupport 327, for example a worm, coupled to an electric motor (notshown) driving the worm in rotation and enabling translation of themagnetic element 326 along the support 327.

In this way, the permanent magnet 324 can rotate and displacevertically.

In use, when it is desired to obtain mixing inside the reaction chamber322, the magnetic generator 325 is operated to generate the rotating andtranslating magnetic field and cause rotation and translation of theferromagnetic anchor 321 inside the reaction chamber 322, as shown inFIGS. 46A and 46B.

As mentioned above, the stirring and mixing group 320 may advantageouslybe used in the system 1 and 1′ according to FIGS. 1-19 . In particular,the microfluidic device 323 may be the cartridge 2 or 2′, the reactionchamber 322 may be the extraction chamber 6, the ferromagnetic anchor321 may be the anchor 97, the magnetic generator 325 may be the anchoractuator 41, the permanent magnet 324 may be the second magnetic element73, the d.c. motor 328 may be the third motor 75, and the support 327may form the second turret 65. Moreover, as explained with reference toFIGS. 1 and 2 , the second turret 65 may be a worm extending vertically,driven in rotation by the second electric motor 72 arranged in the base50 and co-operating with a counter-thread formed on the casing of thethird electric motor 75. The second electric motor 72 is controlled bythe control unit 35.

In this case, for a cartridge 2 or 2′ having dimensions of 75 mm×50mm×10 mm, with the extraction chamber 6 having a volume of approximately1.2 ml and a minimum width of 0.8 mm, the ferromagnetic anchor 321 mayhave a cylindrical shape, with a length of about 7 mm, a diameter ofabout 1.5 mm, and a weight of less than 0.1 g.

The magnetic element 326 may rotate at a maximum nominal speed ofrotation of up to 140 r.p.m., even though the speed in general is notconstant and depends upon the friction with the liquid in the reactionchamber and possible magnetic beads (as described above for thetreatment of the molecules separated in the extraction chamber, withreference to FIGS. 9 and 18A, 18B).

FIGS. 48-49 represent another reliable, rapid, and effective solution toobtain stirring and mixing of a liquid in a microfluidic device. Thissolution may moreover advantageously be used in the cartridges 2 and 2′of FIGS. 1-19 , in addition to and/or instead of the solution describedwith reference to FIGS. 45-47 . Specifically, according to FIGS. 48A and48B, air, preferably filtered, is bubbled into a reaction chamber 331 ofa microfluidic device 330.

In detail, in the microfluidic device 330 of FIGS. 48A and 48B, thereaction chamber 331 is connected to an inlet channel 333, an outletchannel 334, and a vent channel 335. In the shown example, the inletchannel 333 and the outlet channel 334 are arranged near a bottom end ofthe reaction chamber 331, on opposite sides thereof, and are equipped,respectively, with a first valve 336A and a second valve 336B. The ventchannel 335 is connected to a top end of the reaction chamber 331. Afirst air filter 337 may be arranged upstream of the inlet channel 333(typically, on the ventilation line 33 of the control machine 3, 3′ ofFIGS. 1 and 12 ). A second air filter 338 may be arranged on the ventchannel 335, within the cartridge 2, 2′. The first and second filters337, 338 may be filters of the HEPA (High-Efficiency Particulate Airfilter) type. In particular, the first filter 337 has the function offiltering possible pollutants or contaminants upstream of the extractionchamber 6 (FIGS. 1-19 ) and of the reaction chamber 331. The secondfilter 338 has the function of preventing potentially dangerousmaterial, such as parts of viruses, from being released into theexternal environment.

A further valve (not shown) may be provided on the vent channel 335.

In use, initially, the first valve 336A is opened and the second valve336B is closed. Next, a liquid (designated as a whole by 339) isintroduced into the reaction chamber 331 through the inlet channel 333(FIG. 48A). Then (FIG. 48 ), by keeping the first valve 336A open and byapplying a suction pressure of, for example, 5·10⁻² atm to the ventchannel 335, air 340 is returned into the reaction chamber 331 throughthe inlet channel 333. In this way, the air 340 tends to swirl upwards,forming bubbles 341. The bubbles 341 move upwards, causing remixing ofthe liquid 339.

After traversing the entire volume of liquid 339 in the reaction chamber331, the air 340 exits from the reaction chamber 331 through the ventchannel 335. Here, the air 340 is filtered by the second filter 338 andcan thus be discharged towards the outside, without any risk ofcontamination.

At the end of the treatment in the reaction chamber 331, the secondvalve 336B is opened, while the first valve 336A is kept open, to enableoutflow of air and emptying of the reaction chamber 331.

The mixing solution described above is particularly effective whenapplied to the cartridge 2 or 2′ of FIGS. 1-19 . FIG. 49 shows, forexample, application to the cartridge 2′. In particular, themicrofluidic device 330 may form the cartridge 2 or 2′, the reactionchamber 331 may form the extraction chamber 6, the inlet channel 33 maybe formed by the inlet fluidic recess 86, the outlet channel may beformed by the output fluidic recess 88, and the vent channel 335 may beformed by the first vent recess 95. Here mixing air 340 is suppliedthrough the fluidic inlet 11, as indicated by arrow 343, and flows offinto the first vent recess 95 towards the waste chamber 7, 7′ (notvisible) through the first fluidic valve 20, as indicated by the arrow344.

In this way, a very effective system is obtained at very low costs(since it requires only a pumping system and fluidic connections alreadypresent in the systems 1 and 1′ of FIGS. 1-19 ).

For instance, mixing via continuous air bubbling of FIGS. 48A, 48B, and49 is particularly advantageous during the flushing with alcoholicsolutions. In this case, tests performed by the present applicant havedemonstrated that by blowing air in a continuous way at a flow rate of60-70 μl/s for example for two minutes results in a highly effectiveflushing. Furthermore, air bubbling has proven particularly effective ifcarried out in a discontinuous way during detachment of the extractednucleic acids from the magnetic beads, as described above with referenceto FIGS. 9 and 18A and 18B. In particular, during detachment, air can besupplied for 10 s, with a flow rate of 30 μl/s, followed by 10 s ofinterruption.

Hereinafter, possible implementations of a solid-reagent containmentunit are described and may be used in microfluidic devices, such assample analysis cartridges containing molecules to be analyzed, forexample nucleic acids.

In portable microfluidic devices performing analysis of nucleic acidsobtained from biological samples, an area is present, also referred toas analysis chamber, that is loaded both with the nucleic acids (orgeneric molecules extracted from a sample to be analyzed) and reagentsallowing the analysis (referred to hereinafter as assay reagents).

It is convenient for the assay reagents to be preloaded into themicrofluidic device to enable easier use thereof. The term “preloading”indicates the introduction of the reagents into the device duringassembly thereof, i.e., prior to its use. With this strategy, the endoperator during use merely has to introduce the sample to be analyzedinto the device, with one simple operation, without having to preparecomplex reaction mixtures to be introduced into the microfluidic device.

However, many reagents used for biochemical analyses (for example, thereaction mixtures for real-time PCR, which include perishable reagents,such as enzymes and fluorophores) have to be stored at a low temperature(between −20° C. and +4° C.) if in a classic liquid form. It would befar from practical to preload these reagents in liquid form, because thedevice should then be transported and stored at low temperature, withconsequent costs and logistic difficulties. Furthermore, liquid reagentsare difficult to confine, and could thus displace duringtransport/storage, thereby causing problems in the analysis. Thesedisplacement problems would increase further when the device hasmultiple analysis chambers having a common connection prior to start theanalysis. In the latter case, during transport/storage of themicrofluidic device, liquid displacement between the various analysischambers may occur, with consequent mixing of different reagents, whichcould affect the results.

It is, instead, convenient to preload the assay reagents in solid form,i.e., dehydrated, for two reasons. First, the perishable reagents thusbecome stable also at room temperature, since the practically totalabsence of water determines a considerable deceleration of the reactionkinetics, including those of the degradation processes of the reagents.Thus, in this way, the need is avoided to maintain a cold chain for thedevice during entire transport and storage thereof. Furthermore, thesolid reagents are intrinsically more stable also from the “mechanical”standpoint; any displacements of the reagents from their own locationbecome less likely, above all if the analysis chamber or chambers is/aredesigned with an appropriate shape or shapes (as will be describedhereinafter).

The reagents may be introduced into the device already in solid form, orin liquid form and then be dehydrated (for example, via lyophilization)in situ immediately after. Next, the device is assembled in dry,controlled atmosphere to prevent any undesired re-hydration of the solidreagents by air humidity, which would jeopardize both chemical andmechanical stability thereof.

In general, once assembly of the device is concluded with the solidreagents on board, it is sealed within a package at controlledatmosphere that does not allow penetration of humidity from the externalair. Furthermore, albeit using a humidity-proof package, in many casesit is desirable for the containment structure to be resistant tohumidity to reduce further the probability of undesirable re-hydrationof the solid reagents. Such re-hydration could occur accidentally duringtransport/storage, but also while introducing the sample into theportable microfluidic device for use thereof. In this step, in fact, theprotective package is opened, and undesirable re-hydration may occureven in a very rapid way. Moreover, if the sample is processed in themicrofluidic device prior to analysis (for example, if a preventivepurification of DNA/RNA is obtained), the time between opening of thepackage and start of the analysis increases, and thus the probabilitiesof undesirable re-hydration increase.

Finally, it is desirable for the solid reagents not to be able todisplace within the containment structure either during packaging orduring storage and transport, or during handling of the microfluidicdevice when it is used.

FIGS. 50A-50D show manufacturing steps of an embodiment of a unit forcontaining solid, particular dried reagents, hereinafter referred to asreagent unit 350, which satisfies the above requirements.

According to FIG. 50A, the reagent unit 350 is manufactured startingfrom a support 351 bonded, for example glued, to a frame body 352. Inthe embodiment shown in FIGS. 50A-50D, the frame body 352 comprises aframe 353A with rectangular base and a plurality of delimiting walls ordiaphragms 353B, which delimit from each other a plurality of analysiscells 354 (two whereof are visible in FIG. 50A). Alternatively, theframe body 352 may comprise just the frame 353A, as discussedhereinafter with reference to FIG. 58 . The support 351 may be of anymaterial, for example silicon, and form an integrated-circuit chip, andthe frame body 352 may also be of any material, for example moldedpolycarbonate. The analysis cells 354 are thus open on one side and havea base of any shape, for example square or rectangular.

According to FIG. 50A, a holding material 355, for example wax and morein particular a paraffin, is introduced into the cells 354 in the liquidstate. The holding material 355 may be introduced using an automaticpipettor or by hand pipetting, by virtue of its low melting point. Forinstance, paraffin “Paraffin wax,” produced by Sigma-Aldrich, productcode 76228, having a melting point of 44-46° C. and “Paraffin wax”produced by Sigma-Aldrich, product code 327204, having a melting pointof 53-57° C., may be used. Other materials may be used instead of wax,provided that they have a similar behaviour as regards the applicationconsidered and thus:

are inert with respect to the reagents treated in the reagent unit 350;

do not interfere with the reactions taking place in the reagent unit;

have a melting point such as not to interfere with the intended analysisprocesses (as discussed hereinafter with reference to FIGS. 57A-57C);

do not melt during transport/storage; and

have a low volatility in the temperature range of interest.

Preferably, moreover, the holding material 355 has the followingcharacteristics:

it is less dense than the solutions of the treated reagents; and

it is transparent at the wavelengths of interest (if a treatment step,for example detection, of an optical type is provided for).

For instance, in addition to paraffin, other waxes may be used, such asbees wax, or polymers such as polycaprolactone, or solid fats, such ascocoa butter, or a gel, such as hydrogel or organogel.

In general, the holding material 355 is an adhesion material that can beembossed at lower temperatures than its own melting point. For instance,it can be embossed at temperatures lower by 5-10° C. than its ownmelting point. Furthermore, the holding material 355 has a melting pointlower than 62° C., preferably lower than 60° C., even more preferablylower than 58° C.

Then, the holding material 355 is allowed to cool until it solidifies.Next (FIG. 50B), a hot-embossing step is carried out using a first mold357 in order to provide a reagent cavity 359 in each analysis cell 354.

In particular, for the embossing six analysis cells 354 arrangedside-by-side two by two in three rows, the first mold 357 shown in FIG.53A may be used. Here, the first mold 357 comprises six first embossingelements 358 having a projecting frustoconical shape, one for eachanalysis cell 354. For instance, each first embossing element 358 mayhave a base with a diameter of 2.5-3 mm, in particular 2.8 mm, and thegeneratrices of the conical shape may have an angle of approximately 10°with respect to the vertical.

The embossing temperature depends upon the used holding material; inparticular, it is set approximately 5-10° C. lower than the meltingpoint of the material. For instance, in case of paraffin, which, asmentioned, has a melting point of 44°-46° C., the embossing temperatureis chosen in the range 35-40° C., for example 38° C., so as to not causemelting of the holding material, but only softening thereof.

As a result of the embossing operation, the reagent cavity 359 in eachanalysis cell 354 here has a frustoconical shape, delimited by retentionwalls 356 formed by the displaced holding material, and extendsthroughout the thickness of the retention walls 356 (it is a throughcavity).

In FIG. 50C, a liquid reagent 360 (comprising one or more reagentsubstances, according to the application of the reagent unit 350) isadded in the reagent cavities 359 and then (FIG. 50D) dehydrated, forexample lyophilized, in a known manner, to form a dried reagent 361within the reagent cavity 359.

The reagent unit 350 thus prepared (see also FIG. 50E) can then be putin a sealed package that acts also as barrier as regards humidity, forstorage and transport.

During transport and storage of the reagent unit 350, and while openingits package for use, the retention walls 356 may exert an adhesionaction on the dried reagent 361, keeping it in position in the cells andpreventing it from exiting the analysis cells 354.

According to a different embodiment, after dehydration, the reagent unit350 is heated to a temperature close or equal to the melting point ofthe holding material of the retention walls 356. In this situation, asshown in FIG. 51 , the retention walls 356 partially melt to form a sortof crust or plug wall 362 on top of the dried reagent 361. The driedreagent 361 is thus surrounded by a structure (formed by the support351, the retention walls 356 and the plug wall 362) that envelops it onall sides, protects it, and isolates it from the external environment.Since the material of the retention walls 356, and thus of the plug wall362, is inert, does not react with the dried reagent 361 during heating,storage, and analysis.

To improve adhesion of the dried reagent 361 (some dehydrated reagentshave lower properties of adhesion to wax) it is possible to createmechanical retention structures along the retention walls 356, as shownin FIGS. 52A and 52B.

In detail, according to this embodiment, after forming the retentionwalls 356 according to FIGS. 50A and 50B (and thus using the first mold357), these walls 356 are treated so as to form a sort of step or detent365 projecting towards the inside of each reagent cavity 359. To thisend (FIG. 52A), the reagent unit 350 is subject to a second embossingstep, using a second mold 363, for example of the type shown in FIG. 53Bor 53C. In detail, the second mold 363 comprises a plurality of secondembossing elements 364, one for each analysis cell 354, analogously tothe first mold 357. The second embossing elements 364 of the second mold363 are formed by two mold portions: a first mold portion 364A (forexample, having a frustoconical shape) having a minor base, with a firstarea, and a second mold portion 364B projecting from the first moldportion 364A and defining a tip or abutting surface intended to restagainst the support 351 of the reagent unit 350. The second mold portion364B has, for example, a cylindrical shape and a base, with a secondarea smaller than the first area. The first mold portion 364A has thefunction of forming the detent 365, whereas the second mold portion 364Bhas only a stop function during embossing. Thus, the first area of thefirst mold portion 364A is also larger than a cross-section area of thefirst embossing elements 358 of FIGS. 50B and 53A arranged at the samedistance from the abutting surface of the first embossing elements 358.The base area of the second mold portion 364B is smaller than theabutting surface area of the first embossing elements 358 of FIGS. 50Band 53A. For instance, the first mold portion 364A may have a major baseof 3-3.4 mm, in particular 3.2 mm, and a minor base of 2.3 mm-2.7 mm, inparticular 2.5 mm, and the second mold portion 364B may have a base areaof 0.8-1.2 mm, in particular 1 mm. The generatrix of the conical shapeof the first mold portion 364A may have an angle of approximately 10°with respect to the vertical.

In FIG. 53B, the first mold portion 364A of the second mold (designatedby 363′) has a conical shape. In FIG. 53C, the first mold portion 364Aof the second mold (designated by 363″) has a frustopyramidal shape. Thesecond mold portions 364B of the second molds 363′ and 363″ are bothcylindrical. However, the shown shapes are merely exemplary, and othershapes are possible, provided that they have a first, wider, moldportion and a second, narrower, mold portion, to form a recess, and thefirst portion 364A of the second mold 363 has at least one dimensionlarger than the first mold 357 at the same distance from the tipabutting surface of the molds 357, 363. Due to the larger base area ofthe first mold portion 364A at the minor base of the first mold 357 atthe same height, during the second embossing step, the retention walls356 are partially squeezed, and part of the material forms the detent365 extending peripherally towards the inside of the reagent cavity 359(FIG. 52A).

Then (FIG. 52B), the liquid reagent 360 is introduced into the reagentcavity 359, and the step described with reference to FIG. 50D is carriedout, thereby lyophilizing the liquid reagent 360.

The detent 365 thus formed contributes to mechanically blocking thedried reagent 361 and to reliably prevent exit thereof from the reagentcavity 359. Obviously, also in the case of the analysis cell 354 of FIG.52B, it is possible to provide a plug wall 362, as has been describedwith reference to FIG. 51 for obtain sealing towards the outside.

FIGS. 54A and 54B show a variant of the process for forming the reagentcavities 359. In this case, during embossing, a deformable mold 368 isused (FIG. 55 ) having embossing elements 369 with a flexible tip so asto be able to cause deformation and widening during the embossing step.For sake of simplicity, FIGS. 54A and 54B show just one analysis cell354, but the reagent unit 350 may contain any number of analysis cells354, for example six, as described previously.

In detail (FIG. 54A), after introducing the holding material 355 inliquid form and hardening it, the analysis cells 354 are embossed usingthe deformable mold 368 of FIG. 55 . The embossing element 369 here hasa generally frustoconical shape, with a first portion 369A defining themajor base, of a first material, which is harder, for example plastic ormetal, and a second portion 369B protruding from the first portion 369Aand forming the tip or minor base of the embossing element 369, of anelastically deformable material, for example silicone rubber.

In FIG. 54A, the embossing element 359 is introduced into the analysiscell 354 with a pressure such as not to cause deformation of the secondportion 369B of the deformable mold 368. Then a first embossing of theholding material 355 is carried out, and the reagent cavity 359initially has a frustoconical shape.

Then (FIG. 54B), the embossing element 369 is further pressed againstthe support 351, causing deformation and lateral widening of the secondportion 369B of the embossing element 369 transversely to the embossingdirection. Widening of the second portion 369B causes squeezing anddisplacement of the holding material 355 away from the support 351 sothat the reagent cavity 359 assumes an hourglass shape and forms anintermediate neck 370 of minimum area.

The neck 370 here forms a retention structure, which acts on the solidreagent 361 after introducing the liquid reagent and dehydration, as forthe detent 365 of FIG. 52B.

Obviously, also for the analysis cell 354 of FIG. 54B it is possible toprovide a protective plug 362, for sealing towards the outside.

According to the embodiment of FIGS. 56A-56D, initially an adhesive tape366 is applied that can be removed prior to packaging the reagent unit350. In detail, FIG. 56A shows the reagent unit 350 after dehydration,and thus containing the dried reagents 361.

Next (FIG. 56B), the support 351 is attached, for example glued, to theframe body 352 on the top side (the side opposite to the adhesive tape366), thus closing the analysis cells 354 at the top. The support 351may be a silicon chip, for example integrating heaters and/or electricalor electronic components useful during analysis.

In FIG. 56C, the adhesive tape 366 is stripped off. The dried reagents361 do not drop, since they are compact and by virtue of the narrowedopening of the analysis cells 354. In FIG. 56D, the reagent unit 350 isturned over. In this step, the dried reagents 361 may slide towards thesupport 351, but cannot exit the analysis cells 354 due to the narrowedopening of the analysis cells 354, on the side that now faces theexternal environment, due to the presence of the retention walls 356, aswell as, possibly, of the detent 365 (FIG. 52B) or of the neck 370 (FIG.54B). Then, the reagent unit 350 may be packaged. Alternatively, theadhesive tape 366 may not be removed, and forms a further physicalbarrier to the displacement of the dried reagents and to the externalhumidity. In this case, the adhesive tape, if necessary, is removedduring use by the end operator.

In all previous embodiments, the reagent unit 350 is introduced into amicrofluidic device, for example, a portable cartridge, to perform theanalysis. Insertion may be made prior to packaging, i.e., in theassembly step, and the reagent unit 350 is bonded to the microfluidicdevice for example by gluing or mechanical fixing. Alternatively, thereagent unit 350 is introduced into the microfluidic device afteropening the package by the end user who performs the analysis, and isblocked in situ by simple fitting.

FIGS. 57A-57C show steps of re-hydration of the dried reagent 361, forits preparation to perform a biochemical analysis.

In detail (FIG. 57A), when it is desired to perform an analysis ofmolecules, for example nucleic acids, the analysis cell 354 is suppliedwith a sample to be analyzed, in liquid form, designated by 371. After afew seconds, the sample to be analyzed 371 re-hydrates the dried reagent361 (FIG. 57B) to form a reagent-sample mixture 372; the analysis maythen be carried out, according to the provided procedures. Duringanalysis, if so provided (as for real-time PCR), the reagent unit 350may be heated. This heating is at times obtained at a temperature higherthan the melting point of the material of the retention walls 356 (forexample, wax, as discussed above). In this case, the retention walls 356(FIG. 57B) may melt and, due to the lower density of their material ascompared to the reagent-sample mixture 372, the former rises at thesurface of the mixture 372, to form a closing surface 373 that preventsevaporation of the reagent-sample mixture 372. Here, the mixture 372 iscontained between the support 351, the delimiting diaphragms 353, andthe closing surface 373.

In any case, since the material of the retention walls 356 and thus ofthe closing surface 373 (for example, wax) has been chosen according tothe criteria described in detail previously, it does not interfere withthe analysis.

FIG. 58 shows a reagent unit 350 that may be used in the cartridge 2 or2′ of FIGS. 1-19 . The reagent unit 350 of FIG. 58 is of a type withsingle analysis cell 354. Here, the support 351 is formed by the chip 48that integrates the heating and temperature-control element, representedschematically in FIG. 59 by a resistor 374. The frame body 352, herecomprising just the frame 353A, is bonded to the chip 48. The frame 353Adelimits internally a single analysis cell 354, accommodating aretention wall 356 that delimits a bag-shaped reagent cavity 359, whichis the same and of the same size as the analysis opening 84B (FIG. 59 ).The reagent cavity 359 may be obtained, as described with reference toFIGS. 50A-50D, using a mold with a single embossing element similar tothe elements 358 of FIG. 50B, but bag-shaped.

FIG. 59 shows insertion of the reagent unit 350 in the analysis recess84′ of the cartridge 2′ of FIGS. 14-15 , namely, with the reagent cavity359 facing the analysis opening 84B. In practice, the reagent cavity 359and the analysis opening 84B form the analysis chamber 8′.

In this way, when the extracted molecules and the elution liquid are fedto the analysis chamber 8′ (as described with reference to FIGS. 19A and19B), they may mix with the dried reagent (here not shown) contained inthe analysis cell 354 for performing the analysis.

FIGS. 60-64 show a variant of the reagent unit 350 that contains aplurality of analysis cells 354 and may be applied to the cartridge 2 or2′ of FIGS. 1-19 .

In detail, in the reagent unit 350 of FIG. 60 , the frame body 352comprises just the frame 353A and delimits six analysis cells 354defined completely in the wax or other similar holding material. Inpractice, a retention structure 375 is arranged therein and has agenerally parallelepipedal shape, corresponding both to the retentionwalls 356 and to the delimiting diaphragms 353 of FIGS. 50-52, 54, 56 .The reagent cavities 359 are formed in the retention structure 375.Moreover, a fluidic channel 376 is formed in the retention structure375, connects together the reagent cavities 359 and extends on a firstface 375A of the retention structure 375 (FIG. 62 ).

A through hole 377 connects the fluidic channel 376 of the retentionstructure 375 to a second face 375B of the retention structure 375 (FIG.62 ).

The retention structure 375 may be obtained using the mold 378 shown inFIG. 61 . In detail, the mold 378 has six embossing elements 379 with afrustoconical shape, to form the reagent cavities 359; a projection 380for forming the through hole 377; and a projecting structure 381 forforming the fluidic channel 376.

In a not shown manner, the reagent cavities 359 in the retentionstructure 375 may be subject to a second embossing to form teeth similarto the detents 365 of FIGS. 52A-52B. Alternatively, the mold 378 may bemodified as in FIG. 55 to have reagent cavities 359 with the shape shownin FIG. 54B.

The reagent unit 350 of FIG. 60 , after reagent insertion anddehydration, is bonded to the chip 48 (including possible resistors 374)in the manner shown in FIG. 62 , thus with its first face 375A. Then thechip 48 closes the reagent cavities 359 at their major base. To thisend, the process described with reference to FIGS. 56A-56D may, forexample, be used.

The reagent unit 350, thus fixed to the chip 48, may be mounted in acartridge 2″, as shown in FIGS. 63 and 64 . The cartridge 2″ has a basestructure similar to the cartridges 2 and 2′, and thus similar parts aredesignated by the same reference numbers and the different parts aredenoted with prime signs. The cartridge 2″ differs substantially fromthe cartridges 2 and 2′ of FIGS. 4-5 and 13-15 in that the analysisrecess (here designated by 84″, on the first face 80A″ of the body 80″)does not have the through opening 84B, but is closed by an analysis wall384 having a plurality of first through holes 385 and one second throughhole 386. In detail, the recess 84B of FIGS. 63, 64 has shape anddimensions corresponding to those of the reagent unit 350 so that thelatter may be inserted with the second face 375B of the retentionstructure 375 directed toward the wall 384 of the analysis recess 84″.The first holes 385 in the wall 384 of the analysis recess 84″ are equalin number to the reagent cavities 359 (here six) and are arranged so asto face, and be in fluidic connection with, the reagent cavities 359after inserting the reagent unit 350 in the analysis recess 84″. Thesecond through hole 386 in the wall 384 of the analysis recess 84″ isarranged so as to face, and be in fluidic connection with, the throughhole 377 in the retention structure 375.

Moreover, the wall 384 of the analysis recess 84″ has, on the secondface 80B″ of the body 80″ of the cartridge 2″, a first connectionchannel 387 connecting the first through holes 385 to a firstcommunication hole 102″ similar to the holes 102B and 102′ of FIGS. 5-6and 14-15 . The second face 80B″ of the body 80″ of the cartridge 2″further has a second connection channel 388 connecting the secondthrough hole 386 in the wall 384 of the analysis recess 84″ to a secondcommunication hole 100″ similar to the holes 100B and 100′ of FIGS. 5-6and 14-15 .

In practice, the second connection channel 388 enables connection of thereagent cavities 359 to the extraction chamber 6 and thus loading of thereagent cavities 359 with the extracted molecules and the elution liquidin the steps described with reference to FIGS. 10 and 19A, 19B.Furthermore, the first connection channel 387 enables venting of thereagent cavities 359 while they are being loaded with the extractedmolecules and the elution liquid, in the same step.

It is noted that, in this embodiment, the silicon chip, directly facingthe reagent cavities 359, has treated areas that have hydrophobicproperties on the outside of the reagent cavities 359, to be able towithhold the reagent/sample mixture 371 (FIG. 57B). In fact, the chip 48is covered, in a known and not shown manner, by a silicon-oxide layerhaving intrinsic hydrophilic properties. Consequently, when, uponthermal cycles and/or the heating as described with reference to FIG.57C, the walls separating the reagent cavities 359 (formed by theretention structure 375) melt, eliminating the physical barrier betweenthe reagent cavities 359, the hydrophilic areas surrounded by thehydrophobic areas enable the reagent/sample mixture 371 to be held inposition.

For instance, the hydrophobic treatment may be obtained by depositing bylamination an appropriate dry film, for example SINR® manufactured byShin Etsu, and subsequent lithographical defining to remove it from theareas underlying the reagent cavities 359. Alternatively, a non-drymaterial may be used, arranged directly in the desired areas bysilk-screen printing or direct printing using piezoelectric print heads.

It is noted that, in FIGS. 63-64 , the first connection channel 387allows air to exit when the sample to be analyzed is introduced. Thefirst connection channel 387 may be rendered hydrophobic in the mannerreferred to above to prevent the reagent/sample mixture 371 fromexiting. In an alternative embodiment, if a vent channel connects thereaction chamber(s) to the external environment, a filter, for examplean EPA filter, may be provided on the vent to prevent any accidentalexit or contamination of the surrounding environment.

Hereinafter, possible implementations of an analysis unit are described,capable of automatically loading a preset amount of a sample containingmolecules to be analyzed, for use in microfluidic devices, such ascartridges for analysis of nucleic acids.

As is known, in portable microfluidic devices performing analysis ofmolecules, for example nucleic acids obtained from biological samples,it is frequently desirable to be able to automatically mix preciseamounts of reagents with equally precise amounts of samples to beanalyzed.

For instance, in the containment unit 350 described above with referenceto FIGS. 50-64 , enabling preloading of the reagents in dehydrated forminto the containment unit, it is desirable that the sample to beanalyzed is supplied to the analysis cells 354 in an automatic way andin a preset stoichiometric proportion with respect to the preloadedreagents. In this way, it is possible to fully exploit the advantagesprovided by the described containment unit 350, in particular itsconsiderable simplicity of use, the obtainable remarkable reduction inthe handling time and minimization of errors.

In general, it is desirable to have a method for mixing of presetamounts of a liquid (for instance, a primary biological sample or apre-treated biological sample) with solids (typically, dehydratedreagents) preloaded in a controlled amount in an analysis cell.

FIGS. 65A-65D refer to a simplified embodiment of an analysis unit 390and of a method for loading samples. In particular, these figures referto the loading of a treated sample containing extracted nucleic acidsand an elution liquid, into an analysis cell containing reagentsspecific for the desired analysis reaction (hereafter referred to as“assay-specific reagents”), for example, for analysis of DNA, withoutthe present disclosure being limited to this application. FIGS. 65A-65Dshow the analysis unit 390 in the use position; thus the indicationssuch as “up,” “down,” “top,” and “bottom” and the like refer to theshown use position.

In detail, as shown in FIG. 65A, the analysis unit 390 comprises ananalysis body 394 accommodating a first chamber 391 and a second chamber392; the first and second chambers 391, 392 are connected together by asupply channel 393 extending also in the analysis body 394.

The first and second chambers 391, 392 have respective inlets 391A, 392Aarranged near the respective top ends, and the first chamber 391 has anoutlet 391B arranged near a bottom end thereof. The supply channel 393here extends between the outlet 391B of the first chamber 391 and anoutlet end 397 of the supply channel 393. Furthermore, the supplychannel 393 has a branch 393A connected to the inlet 392A of the secondchamber 392. The branch 393A here extends vertically. The inlet 391A ofthe first chamber 391 is connected to an inlet 398 of the analysis unit390.

A valve 405 may be provided on the supply channel 393.

It is noted that, in a manner not shown, a vent channel may be provided,connected to the second chamber 392 to let out air when the sample to beanalyzed is introduced. The vent channel may be rendered hydrophobic toprevent also liquid from coming out. If the vent channel is connected tothe external environment, to prevent any accidental exit orcontamination of the surrounding environment, a filter, for example anEPA filter, may further be provided on the vent channel.

The second chamber 392 contains dried reagents 395, for example amixture of reagents for performing real-time PCR, previously preloaded,in particular during the step described with reference to FIGS. 50C-50Dand on the basis of the methodology described below. The first chamber391 contains a sample to be analyzed 396 in liquid form, for example abiological sample or a derivative thereof, in particular the extractednucleic acids and the elution liquid, as described with reference toFIGS. 1-19 . The sample to be analyzed 396 is generally in excess withrespect to the amount to be mixed with the dried reagents 395; theamount thereof might not be known precisely. The second chamber 392 thusforms an analysis chamber, where analysis of the sample to be analyzed396 is carried out.

In FIG. 65B, a first force Fl is applied. The first force Fl may beexerted, as a thrust force, by an external pump (not shown), connectedto the inlet 398 of the analysis unit 390, and/or may be a passive force(for example, exploiting capillarity) acting from the outlet 391B and/ormay derive from a negative pressure (suction pressure) applied on theoutlet end 397 of the supply channel 393 and transferred to the firstchamber 391 through the supply channel 393.

The first force Fl causes the sample to be analyzed 396 to exit from thefirst chamber 391 through the outlet 391B thereof and fill the supplychannel 393. By capillarity, the sample to be analyzed 396 also entersthe second chamber 392.

When the sample to be analyzed 396 penetrates into the second chamber392, it comes into contact with the dried reagents 395, which start toabsorb it by hydrophilia (FIG. 65C), to form a sample/reagent mixture399. The amount of sample to be analyzed 396 entering the second chamber392 depends upon the amount of liquid that can be absorbed by the driedreagents 395 and is predetermined at the design stage, as discussed indetail hereinafter.

If so desired (FIG. 65D), the first chamber 391 and the supply channel393 may be emptied by applying a second force F2 on the outlet end 397of the supply channel 393. Alternatively, the second force F2 may beapplied at the inlet 398 of the analysis unit 390. The second force F2may be an active force, for example a positive or negative pressuregenerated by an external pump. The second chamber 392 is not emptied.

The analysis unit 390 of FIGS. 65A-65D may advantageously be used withthe cartridge 2, 2′ described with reference to FIGS. 1-19 and with thecontainment unit 350 described with reference to FIGS. 50-59 . Inparticular, the first chamber 391 may be formed by the extractionchamber 6 (FIGS. 1-19 ), the second chamber 392 may be formed by thecollector 8, 8′ or by the analysis cell 354 in the reagent unit 350 ofFIGS. 58-59 , and the inlet 398 of the analysis unit 390 corresponds tothe inlet fluidic recess 86. In this case, the analysis body 394 isformed by a number of parts, i.e., by the bodies 80-82 or 80′-82′, whichhouse the first chamber 391 (FIGS. 4 and 13 ), and by the reagent unit350, accommodating the second chamber 392. The supply channel 393 may beformed by the output fluidic recess 88, by the product recess 99, 99′,and by the through holes 100A, 100′.

FIG. 66A shows a different embodiment of the analysis unit 390. Indetail, the analysis unit 390 of FIG. 66A differs from the analysis unit390 of FIG. 65A in that it comprises a plurality of second chambers orwells 392 ¹, 392 ², 393 ³, . . . , 392 ^(n), connected to the supplychannel 393 through respective branches 393 ¹, 393 ², 393 ³, . . . , 393^(n).

Different dried reagents 395 ¹, 395 ², 395 ³, . . . , 395 ^(n) may bepreloaded in the second chambers 392 ¹, 392 ², 393 ³, . . . , 392 ^(n).In this way, the analysis unit 390 can carry out different reactionsstarting from a same sample to be analyzed 396.

In FIG. 66B, the second chambers 392 ¹, 392 ², 393 ³, . . . , 392 ^(n)are loaded with the sample to be analyzed 396 contained in the firstchamber 391; they are loaded in sequence on the basis of theirarrangement along the supply channel 393, i.e., according to theirdistance from the first chamber 391, by applying the first force F1.

After loading the sample to be analyzed 396 (FIG. 66C), the supplychannel 393 may be emptied by applying a second force F2, of an activetype. A different sample/reagent mixture 399 ¹, 399 ², 399 ³, . . . ,399 ^(n) is thus contained in each second chamber 392 ¹, 392 ², 393 ³, .. . , 392 ^(n) present.

Then (FIG. 66D), the second chambers 392 ¹, 392 ², 393 ³, . . . , 392^(n) may be isolated from each other using an inert liquid non-mixablewith the sample/reagent mixture 399, designated by 400, for example byloading mineral oil or liquid paraffin, or by melting some material,such as low-melting paraffin wax, forming the walls of the secondchambers 392′, 392″, 392′″, . . . , 392 ^(n), in a not shown manner.

The analysis unit 390 of FIGS. 66A-66D may be applied to the cartridge2″ of FIGS. 60-64 using the containment unit 350 described withreference to FIGS. 50-57C. In this case, the first chamber 391 may beformed by the extraction chamber 6 (extraction recess 83), the secondchambers 392 ¹, 392 ², 393 ³, . . . , 392 ^(n) correspond to theanalysis cells 354 or to the reagent cavities 359, and the supplychannel 393 corresponds to the fluidic channel 376 of FIG. 60 , to thethrough hole 377, and to the recesses and holes 88, 388, and 100″ ofFIGS. 63-64 . In addition, in this case, for example, isolation betweenthe second chambers 392 ¹, 392 ², 393 ³, . . . , 392 ^(n) may beobtained by melting the retention structure 375.

FIG. 67 is a schematic illustration of an analysis unit 390 having adifferent arrangement of the second chambers 392. Here, moreover, thefirst chamber 391 is arranged in a separate unit 410. The analysis unit390 of FIG. 67 has six second chambers 392 ¹, 392 ² 392 ³, . . . , 392⁶, even though the number may vary, and they are arranged in twovertical rows. The supply channel 393 thus has two branchesseries-connected, extending vertically, one of them defining an inlet403 and the other defining an outlet 404 of the analysis unit 390.

Here, the second chambers 392 ¹, 392 ² 392 ³, . . . , 392 ⁶ areconnected to the supply channel 393 through branches 393 ¹, 393 ², 393³, , 393 ⁶ with horizontal extension.

For the rest, the analysis unit 390 is similar to the analysis unit 390of FIGS. 65 and 66 , and loading of the sample to be analyzed 396 (herenot represented) is the same as described above with reference to thesefigures.

For instance, the analysis unit 390 may be formed by the reagent unit350 of FIGS. 60-64 , and the separate unit 410 may be formed by thecartridge 2″ shown in FIGS. 63 and 64 .

In all of FIGS. 65-66 , the second chambers 392, 392 ¹-392 ^(n) may havea volume of for example 5-30 μl.

Obviously, the arrangement and the number of second chambers 392 ¹-392^(n), their volume, their connection to the supply channel 393, andtheir sequence on the supply channel 393 may vary as desired, accordingto the need.

To absorb the liquid sample to be analyzed, the capillarity of thebranch or branches 393A, 393 ¹, . . . , 393 ^(n) is exploited, asmentioned above. The dimensions (radius and length) of these branches,and possibly also of the supply channel 393 (if it is desired to useonly the capillarity as force F1) are appropriately calculated,according to the criteria referred to below.

For an estimate of the order of magnitude, Jurin's law is used, whichdescribes the height of the meniscus of a liquid in a capillary tube,the top opening whereof is exposed to some known pressure and whereingravity counters the rise of the meniscus (worst case). Even though theconditions of application are different, with some approximations it ispossible to obtain a rough size estimate during design of the branches393A, 393 ¹, . . . , 393 ^(n) and possibly of the supply channel 393.

In this estimate, the pressure of thrust or suction (force F1 of FIGS.65B, 66A) is neglected, and it is assumed that the pressure in thecapillary tube is constant.

In these conditions, the height of the liquid is given by:

$\begin{matrix}{h = \frac{2\gamma\cos\theta}{\rho{gr}}} & (1)\end{matrix}$

where γ is the surface tension (in J/m² or N/m), θ is the contact anglebetween the surface of the liquid and the wall of the supply channel393/393 ¹, . . . , 393 ^(n), ρ is the density of the liquid, g is theacceleration of gravity, and r is the radius of the supply channel393/393 ¹, . . . , 393 ^(n).

If the liquid is water, Eq. (1) becomes:

$h_{H_{2}O} = {\frac{1.48 \times 10^{- 5}}{r}\lbrack m\rbrack}$

According to this model, with a supply channel 393/393 ¹, . . . , 393^(n) of radius r=1 mm, the height of the meniscus, and thus the usefullength of the supply channel 393/393 ¹, . . . , 393 ^(n) for exploitingthe capillarity is approximately 1.5 cm. This value represents in anycase a rough estimate, and the design of the analysis unit 390 is madeby including an empirical characterization.

In particular, in case of the analysis unit 390, the supply channel393/393 ¹, . . . , 393 ^(n) has a rectangular cross-section, typicallywith a base of 1 mm and a height of 0.5-1 mm. The maximum length of thesupply channel 393/393 ¹, . . . , 393 ^(n) is thus in the region of afew centimeters, compatible with the dimensions of the analysis unit390, which allows just the force of capillarity (if so desired) to beexploited in the type of device here considered.

According to one aspect of the present disclosure, in all the analysisunits 390 shown, the reaction reagents 395 in the second chambers 392are contained in an alveolar reaction mass.

The alveolar reaction mass has a roughly spongy structure and has theaim of:

helping the dried reagents to remain in position during transport andstorage of the analysis unit 390;

enabling loading of predetermined amounts of sample to be analyzed (theso-called “self-aliquoting”); and

preventing or at least reducing cross-contamination between differentdried reagents in different chambers and wells after re-hydrating thedried reagents with the sample to be analyzed, which is possible byvirtue of the connection between the various chambers or wells.

For instance, when the analysis unit 390 is formed by or is incorporatedin the cartridge 2, 2′ or 2″, the alveolar mass adheres to the support351 and remains in position even after the retention structure 356, 375has melt, as described with reference to FIGS. 57C and 60-65 .

Because of the presence of the alveolar reaction mass, in the designstage the volume of the second chamber or chambers 392 is calculatedtaking into account not only the amount of sample to be analyzed that isto be absorbed, but also the possible swelling of the alveolar mass.

The alveolar reaction mass enables absorption of a preset amount ofsample to be analyzed, provides greater stability to the dried reagents,holding them within the second chamber or chambers, and favoursattraction of the sample to be analyzed, in liquid form.

This is all the more useful when the analysis unit 390, 390′ forms thereagent unit 350 shown in FIGS. 63, 64 , where the retention structure375 melts during the thermal cycles. In this way, in fact, each reactionsite is precisely defined, ensuring repeatability of the reaction,proper control of temperature, and correct analysis (detection step).

The alveolar reaction mass is typically obtained by lyophilization,which includes steps of freezing, primary drying and secondary dryingthe assay-specific reagents.

The alveolar mass is formed by one or more excipients having the aim offorming a matrix that receives the reagents to be dehydrated. Theexcipients are, for example, chosen in the group comprising: agarose,calcium alginate, polyacrylamide, hydroxyethyl cellulose, polyethyleneglycol, and zeolites. In general, the excipient or excipients inquestion meets/meet the following requisites:

stable structure both during re-hydration and possibly as thetemperature varies;

limited re-swelling; and

-   -   hydrophilia, and more precisely capacity to absorb both the        assay reagents and the sample.

The amount of liquid (sample to be analyzed) entering the secondchambers 392 and absorbed by the alveolar mass depends upon:

the concentration of the excipient forming the alveolar mass in theinitial solution that is lyophilized; the greater the amount ofexcipient, the greater the amount of dehydrated molecules of theexcipient that can undergo hydration with the sample to be analyzed; inaddition, as the amount of the excipient increases, the resistance ofthe alveolar mass increases, the alveolar mass can thus absorb a greateramount of sample to be analyzed without dissolving;

the degree of crosslinking, if the excipient is a polymer capable ofcrosslinking; crosslinked polymers are in general stiffer, and this factmay be useful in this application; the increase in the degree ofcrosslinking and thus of the stiffness of the alveolar mass enables thelatter to absorb the sample to be analyzed without dissolving and with alower re-swelling;

the ratio between the volume of the analysis chamber and the volume ofthe dried excipient: if the alveolar mass swells during re-hydration,this ratio becomes important; in fact, the absorption of liquid (sampleto be analyzed) is interrupted when the alveolar mass occupies theentire volume of the reaction chamber during re-hydration.

It follows that, once the three parameters referred to above, which canbe controlled, are fixed, the amount of sample to be analyzed that canbe absorbed by the alveolar mass becomes “stoichiometric” in a preciseand repeatable way.

The above amount may be calculated empirically by performing anexperiment using a video camera with high frame rate and highresolution, so as to film re-hydration, step after step (for example, byadding 1 μl at each step), of the dried alveolar mass. When the alveolarmass stops absorbing the liquid sample, a part of the liquid starts toform a “shell” that surrounds the alveolar mass, and possibly thisstarts to lose its own shape (according to the characteristics of themass). It has been shown by experiments of the present applicant thatthese phenomena are clearly visible and enable the exact amount ofsample to be analyzed absorbed by the alveolar mass to be determined.

For instance, an alveolar mass obtained by lyophilization of 20 μl of anaqueous solution of agarose at 4% of mass of solute per volume ofsolution (w/V) absorbs 15 μl of water in a reaction chamber having avolume of 21.5 μl.

The alveolar mass may be produced using a multi-stage lyophilizationprocess.

For instance, and in a non-limiting way, the process for producing thealveolar mass may comprise two steps:

1. first lyophilization of a solution containing the excipient orexcipients and possible lyoprotectants (i.e., molecules that, combinedwith the excipients, prevent or substantially reduce chemical andphysical instability of the reagents that are introduced in thesubsequent step 2a during their lyophilization and subsequent storage);for instance, sugars, amino acids, methylamines, etc. may be used aslyoprotectants; in this step, an intermediate alveolar mass is formed;and

2a. introduction of a solution of assay-specific reagents, for example amixture of real-time-PCR-specific reagents plus possible lyoprotectants,in the intermediate alveolar mass, and re-hydration of the excipient orexcipients (plus possible lyoprotectants) lyophilized/obtained in step 1by the assay-specific reagents (plus possible lyoprotectants); and

2b. second lyophilization.

The first lyophilization may comprise, for example, four sub-steps:

a) preparation of a liquid solution of a precursor of the desiredexcipient (including possible lyoprotectants) monomeric or already inthe polymeric form; for instance, an aqueous solution of agarose may beprepared, with a concentration of 2-10% in mass of solute per volume ofsolution (w/V);

b) freezing at a temperature of −40° C. to −80° C., for two hours;

c) primary drying (sublimation) for a time of 6-24 hours, at a very lowpressure, for example 0.1 mbar; and

d) secondary drying (desorption), which may last up to half of theduration of the previous step c). The secondary drying may be carriedout, for example, at the pressure of 0.1 mbar by heating the plates ofthe lyophilizer at 30° C.

The second lyophilization (2b) may be carried out in a similar manner towhat described for the first lyophilisation.

At the end of the second lyophilization, an alveolar mass is obtained,which incorporates the assay-specific reagents.

The alveolar mass thus obtained may be introduced into the secondchambers 393, 393 ¹-393 ^(n); it is able to absorb a precise volume ofre-hydration liquid (sample to be analyzed), as explained above.

Lyophilization in two separate steps is particularly advantageous sinceit enables maximum freedom of choice of the excipients (plus possiblelyoprotectants) and of the reagents for the analysis, which may bedeveloped, produced, and purchased independently, using protocols notshared between the manufacturer of the cartridge and the assaymanufacturer. Furthermore, it enables a high final concentration (bothof the assay-specific reagents and of the excipients plus possiblelyoprotectants) to be obtained with values that cannot be achieved in asingle lyophilization step.

As an alternative to the above, in some applications and for some assayreagents it is possible to carry out just one lyophilization whereinboth the excipients intended to form the alveolar mass and theassay-specific reagents are dehydrated simultaneously.

When the reaction unit 390 forms the cartridge 2 or 2′ of FIGS. 4-6 and13-15 , the intermediate alveolar mass or the alveolar mass may beproduced directly within the analysis chamber 8, 8′, prior to coupling,for example bonding or force fitting the body 80, 80′ in the secondclosing wall 82, 82′. To this end, first the liquid solution of theexcipient (as resulting from step a) described above) or the liquidsolution of the assay-specific reagents is loaded in the analysischamber 8, 8′ using an automatic pipettor or by hand pipetting, and thenlyophilization is carried out.

For the reaction unit 390, formed by the containment unit 350 of FIGS.50-64 , lyophilization(s) may be performed directly in the containmentunit 350.

With the solution of FIGS. 64-66 , it is possible to obtain aninexpensive and precise loading of a sample to be analyzed in ananalysis chamber containing dried reagents in a simple way. The presenceof the dried reagents in alveolar form enables loading to be obtained inprecise amounts. The possible treatment to obtain the dried reagents inalveolar form using two lyophilization steps enables an increase in theconcentration of dried reagents (excipient or excipients, possiblelyoprotectants, and reagents for the analysis) and thus enables a highchemical and physical stability of the alveolar mass and a higheranalysis efficiency. Furthermore, the described solution enables themanufacturer of the cartridge 2, 2′, 2″ and the assay manufacturer notto share their own know-how, which is at times not public.

According to yet another aspect of the present disclosure, when thecartridge 2, 2′ of FIGS. 1-19 integrates an analysis chamber 8, 8′wherein the analysis of the treated sample is carried out as describedabove, as in the case of the cartridge 2″ of FIGS. 63-64 , the controlmachine 3, 3′ is able to identify and automatically handle the intendedtype of analysis, as described hereinafter. It is noted that, eventhough hereinafter, for sake of simplicity, reference is made to thecartridge 2 and to the control machine 3, the following explanation alsoapplies to the cartridge 2′ of FIGS. 13-19 and to the cartridge 2″ ofFIGS. 63 and 64 , as well as to the machine 3′ of FIGS. 11-12 .

In particular, to enable automatic handling of the analysis, dataregarding the analysis for which the cartridge 2 is designed are storedon the cartridge 2, also considering the specific reagents for thesample contained in the analysis chamber 8.

To this end (FIG. 68 ), the machine 3 comprises a radiofrequency antenna410 coupled to the control unit 35, and the cartridge 2 has an RFID(Radio-Frequency

Identification) tag 411. The RFID tag 411 is typically of a passive typeand is arranged on the casing 5 or co-molded with the casing 5 andcomprises an antenna and a writing substrate, as known (and not shown).

In particular, the RFID tag 411 contains information on the type ofcartridge 2, including:

number of analysis chambers or wells 8, 354, 359 contained in thecartridge 2 or in the containment unit 350/analysis unit 390 of FIGS.50-67 ;

type of analysis to be performed in the cartridge 2;

expiry date of the cartridge 2; and

traceability data, regarding, for example, production andfunctionalization of the cartridge 2.

The RFID tag 411 may be read by the control machine 3, using its ownradio-frequency antenna 410 or using a mobile device 412, for example acellphone, through a common NFC (Near-Field Communication) interface.

Typically, the RFID tag 411 interacts with the control machine 3 beforeand after performing an analysis; it interacts with the mobile device412 after performing an analysis, as represented in the flow charts ofFIGS. 69 and 70 .

In particular, when it is desired to carry out an analysis (FIG. 69 ),the cartridge 2 is inserted in the control machine 3 (step 415), thecontrol machine 3 reads the information stored in the RFID tag 411,referred to above (step 416), and, on the basis of the information read,the control machine 3 is able to start the intended operations for thecorrect type of analysis, which include the sample preparation (forexample, coupling of containers 46 in the desired sequence, activatingthe actuators 40-43, the pump 25, the heaters 48, etc., as describedwith reference to FIGS. 7-10 , or similar operations described withreference to FIGS. 16-19 ). Then, the machine 3 may perform the properanalysis operations, and read the results, in a per se known manner.

After analysis (FIG. 70 ), the control machine 3 sends the results ofthe analysis to the cartridge 2 through its own antenna 410 (step 420).The cartridge 2 receives and writes these results through its own RFIDtag 411 (step 421). These data may then be read at any moment, forexample via the cellphone 412 having an NFC (Near-Field Communication)protocol (step 422).

In this way, the results of the analysis may be stored and read afterquite some time, facilitating handling of the stored data. In fact, ifthese data were stored for example in the cloud, access might be farfrom practical. For instance, due to the large number of performedanalyses, the identifiers, provided to the users, would be very long andthus far from practical to use.

The data could be protected by encryption algorithms, to safeguard theprivacy of the patients.

Finally, it is clear that modifications and variations may be made tothe solutions described and illustrated herein, without therebydeparting from the scope of the present disclosure, as defined in theattached claims. For instance, the various described embodiments may becombined to provide further solutions.

The various embodiments described above can be combined to providefurther embodiments. These and other changes can be made to theembodiments in light of the above-detailed description. In general, inthe following claims, the terms used should not be construed to limitthe claims to the specific embodiments disclosed in the specificationand the claims, but should be construed to include all possibleembodiments along with the full scope of equivalents to which suchclaims are entitled. Accordingly, the claims are not limited by thedisclosure.

1. A method for sample preparation and molecule analysis, comprising:introducing a portable microfluidic device into a support; introducing asample into a sample inlet opening of the portable microfluidic device;extracting molecules from the sample to obtain an analyzable sample;transferring the analyzable sample into an analysis volume of a solidreagent containment unit of the portable microfluidic device;re-hydrating a solid reagent in the solid reagent containment unit withthe analyzable sample; and conducting an analysis on the analyzablesample, wherein the portable microfluidic device includes: an extractionchamber in fluidic connection with an opening for introducing samples; awaste chamber extending along the extraction chamber; a collectorextending along the extraction chamber and the waste chamber; a fluidiccircuit connecting the extraction chamber, the waste chamber, and thecollector; and the solid reagent containment unit which includes: asupport; a frame body fixed to the support, the frame body delimitinginside, together with the support, an analysis volume; areagent-adhesion structure within the analysis volume; and a reagentcavity, which extends within the reagent adhesion structure, wherein:the reagent adhesion structure is formed by an inert adhesion materialembossable at lower temperatures than a melting point of the adhesionmaterial, the reagent cavity houses solid reagents, and the collectorcomprises an analysis recess designed to house the solid reagentcontainment unit, with the analysis recess in fluidic connection withthe reagent cavity.
 2. The method for sample preparation and moleculeanalysis of claim 1, wherein the support of the solid reagentcontainment unit is an integrated circuit chip.
 3. The method for samplepreparation and molecule analysis of claim 1, wherein integrated circuitchip includes a heater that is aligned with the reagent cavity of thesolid reagent containment unit
 4. The method of claim 1, wherein:transferring the sample into the analysis volume of the solid reagentcontainment unit of the portable microfluidic device further includes:opening a valve to open a fluidic pathway to the analysis chamber alongwhich the analyzable sample travels along to the analysis chamber; andgenerating suction pressure with a pump in fluid communication with thetransportable microfluidic device; re-hydrating the solid reagent in thesolid reagent containment unit with the analyzable sample furtherincludes introducing the analyzable sample to the reagent cavitycontaining the solid reagent.
 5. The method of claim 1, whereinextracting molecules from the sample to obtain the analyzable sampleincludes: introducing a preparation reagent into the extraction chamberin which the sample and magnetic beads are present by activating anlight emitting diode (LED) opening a one-shot valve along an inletfluidic recess of the transportable microfluidic device, by opening afirst valve hole through deforming a first shutter, and by generating asuction pressure with a pump; activating an anchor actuator moving androtating an anchor in the extraction chamber to stir and mix thepreparation reagent, the sample, and the magnetic beads in theextraction chamber extracting the molecules from the sample and adheringthe magnetic beads to the molecules; removing the preparation reagentsfrom the extraction chamber while leaving the magnetic beads and themolecules within the extraction chamber by opening a second valve holethrough deforming a second shutter and activating a blocking actuatorattracting the magnetic beads and holding the magnetic beads to whichthe molecules are adhered within the extraction chamber; flushing themolecules by introducing in at least one flushing liquid into theextraction chamber; and forming the analyzable sample by introducing anelution liquid purifying the molecules and separating the molecules fromthe magnetic beads.
 6. The method of claim 5, wherein removing thepreparation reagent from the extraction chamber includes introducing thepreparation reagent to the waste chamber within the transportablemicrofluidic device delimited by the first wall, the second wall, andthe body.
 7. The method of claim 5, wherein flushing the molecules byintroducing the at least one flushing liquid into the extraction chamberfurther includes introducing a plurality of flushing liquids insuccession to the extraction chamber.
 8. The method of claim 1, whereinthe molecules are a nucleic acid present within the sample.
 9. A method,comprising: introducing a sample into an extraction chamber of aportable microfluidic device by introducing the sample into a sampleinlet opening of the portable microfluidic device, the extractionchamber being defined by a first wall of the portable microfluidicdevice, a second wall of the portable microfluidic device, and a body ofthe portable microfluidic device that is between the first wall and thesecond wall; extracting molecules from the sample in the extractionchamber obtaining an analyzable sample; transferring the analyzablesample to an analysis chamber of the portable microfluidic devicedelimited by the body and the first wall; exposing the analyzable sampleto a solid reagent in a reagent cavity of a reagent unit and on anintegrated circuit chip of the reagent unit, the reagent cavity beingdelimited by a sidewall of an embossable holding material of the reagentunit and a surface of the integrated circuit chip; and analyzing theanalyzable sample exposed to the solid reagent within the reagentcavity.
 10. The method of claim 9, wherein extracting molecules from thesample in the extraction chamber obtaining the analyzable sampleincludes: introducing a preparation reagent into the extraction chamberin which the sample and magnetic beads are present by activating anlight emitting diode (LED) opening a one-shot valve along an inletfluidic recess of the portable microfluidic device, by opening a firstvalve hole through deforming a first shutter, and by generating asuction pressure with a pump; activating an anchor actuator moving androtating an anchor in the extraction chamber to stir and mix thepreparation reagent, the sample, and the magnetic beads in theextraction chamber extracting the molecules from the sample and adheringthe magnetic beads to the molecules; removing the preparation reagentsfrom the extraction chamber while leaving the magnetic beads and themolecules within the extraction chamber by opening a second valve holethrough deforming a second shutter and activating a blocking actuatorattracting the magnetic beads and holding the magnetic beads to whichthe molecules are adhered within the extraction chamber; flushing themolecules by introducing at least one flushing liquid into theextraction chamber; and forming the analyzable sample by introducing anelution liquid purifying the molecules and separating the molecules fromthe magnetic beads.
 11. The method of claim 10, wherein transferring theanalyzable sample to the analysis chamber of the portable microfluidicdevice delimited by the body and the first wall further includes openinga third valve hole along a fluidic pathway that extends from theextraction chamber to the analysis chamber by deforming a third shutterand generating the suction pressure with the pump.
 12. The method ofclaim 10, wherein removing the preparation reagent from the extractionchamber includes discharging the preparation reagent from the portablemicrofluidic device through a fluidic outlet of the portablemicrofluidic device.
 13. The method of claim 10, wherein removing thepreparation reagent includes introducing the preparation reagent to awaster chamber within the portable microfluidic device delimited by thefirst wall, the second wall, and the body.
 14. The method of claim 10,further comprising heating the solid reagent with a heater of theintegrated circuit aligned with the reagent cavity in which the solidreagent is present.
 15. A method, comprising: introducing a sample intoan extraction chamber of a portable microfluidic device dissolving atablet present within the extraction chamber releasing a plurality ofmagnetic beads within the extraction chamber, the extraction chamberbeing defined by a first wall of the portable microfluidic device, asecond wall of the portable microfluidic device, and a body of theportable microfluidic device that is between the first wall and thesecond wall; extracting molecules from the sample in the extractionchamber obtaining an analyzable sample including: introducingpreparation reagents within the extraction chamber in which the sampleis present; activating an anchor actuator to rotate an anchor within theextraction chamber to stir and mix the sample with the preparationreagents extracting the molecules from the sample and adhering themolecules to the magnetic beads; magnetically attracting the magneticbeads towards a blocking actuator by activating the blocking actuatorto; and removing the preparation reagent spent from extracting themolecules from the sample; transferring the analyzable sample to ananalysis chamber of the portable microfluidic device delimited by thebody and the first wall; exposing the analyzable sample to a solidreagent in a reagent cavity of a reagent unit, the reagent cavity beingdelimited by a sidewall of an embossable holding material of the reagentunit and a surface of an integrated circuit chip of the reagent unit;and analyzing the analyzable sample exposed to the solid reagent withinthe reagent cavity.
 16. The method of claim 15, wherein extracting themolecules from the extraction chamber obtaining the analyzable samplefurther includes: introducing a preparation reagent into the extractionchamber in which the sample and magnetic beads are present by activatingan light emitting diode (LED) opening a one-shot valve along an inletfluidic recess of the portable microfluidic device, by opening a firstvalve hole through deforming a first shutter, and by generating asuction pressure with a pump; flushing the molecules by introducing atleast one flushing liquid into the extraction chamber; and forming theanalyzable sample by introducing an elution liquid purifying themolecules and separating the molecules from the magnetic beads.
 17. Themethod of claim 16, wherein flushing the molecules by introducing the atleast one flushing liquid into the extraction chamber further includesintroducing respective liquids of a plurality of flushing liquids intothe extraction chamber.
 18. The method of claim 17, wherein introducingthe respective liquids of the plurality of flushing liquids into theextraction chamber further includes introducing successively therespective liquids of the plurality of flushing liquids into theextraction chamber
 19. The method of claim 18, wherein the respectiveliquids of the plurality of flushing liquids are stored within aplurality of reagent chambers within the portable microfluidic device,and the plurality of reagent chambers are delimited by the first wall,the second wall, and the body.
 20. The method of claim 18, wherein themolecules are a nucleic acid present within the sample.